Modification of Ribonuclease A Induced by 2-Chlorocyclohexa-2,5 … · 2017-10-10 · 2 ABSTRACT...

1

Modification of Ribonuclease A Induced by

2-Chlorocyclohexa-2,5-diene-1,4-dione

by

Albert R. Vaughn

Departmental Honors Thesis

The University of Tennessee at Chattanooga

Department of Chemistry

Project Director: Dr. Jisook Kim

Examination Date: March 16, 2010

Committee Members:

Dr. Jisook Kim

Dr. Tom Rybolt

Dr. John Lynch

Dr. Kathleen Wheatley

Project Director

Department Examiner

Department Examiner

Liaison, Department Honors Committee

Chairperson, University Departmental Honors Committee

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ABSTRACT

Due to their reactivity, polycyclic aromatic hydrocarbon (PAH) metabolites can modify

cellular components. One such PAH metabolite is 2-Chlorocyclohexa-2,5-diene-1,4-dione.

Presented here are the effects ClpBQ has on individual amino acids and the model protein RNase

A. The individual amino acids evaluated in this study were lysine and cysteine. Kinetic data

was elucidated for the reaction between ClpBQ and lysine. The rate constant for this reaction is

0.2258 min-1

± 0.02133. We attempted to find the rate constant for the reaction between ClpBQ

and cysteine, but we were not successful; however it was determine that a reaction did occur.

UV/Vis spectral data from DNP trapping has shown lysine is oxidized to an aldehyde by ClpBQ.

With regard to RNase A, SDS-PAGE data suggests ClpBQ induces protein crosslinking and very

likely adduct formation. The degree of RNase A modification is increased by both an increase in

the concentration of ClpBQ in solution and an increase in the time of incubation. Furthermore,

this project examined two common reducing agents and discovered both NADH and L ascorbic

acid can inhibit RNase A modification by ClpBQ, but inhibition of protein modification by L

ascorbic acid is more efficient.

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TABLE OF CONTENTS

Cover Page ……………………………………………………………………………………….1

Abstract …………………………………………………………………………………………..2

Table of Contents ………………………………………………………………………………..3

Glossary ………………………………………………………………………………………….4

Chapter 1: Introduction ………………………………………………………………………….5

Chapter 2: Chemical Model Reactions …………………………………………………………12

2.1 Background ………………………………………………………………………….13

Chapter 2 References …………………………….………………………………17

2.2 Results and Discussion ………………………………………………………………18

2.3 Conclusions …………………………………………………………………………21

Chapter 2 Figures …………………………………………..……………………22

2.4 Experimental ………………………………………………………………………32

Chapter 2 Tables ………………………………….…………..…………………34

Chapter 3: Protein Model Reactions …………………………………………..………………35

3.1 Background ………………………………………………………….………………36

Chapter 3 References ……………………………………………………………42

3.2 Results and Discussion ………………………………………………………………43

3.3 Conclusions ……………………………………………….…………………………50

Chapter 3 Figures ……………………………………..…………………………51

3.4 Experimental …………………………………………………………………………61

Chapter 3 Tables …………………………………………………………………68

Appendix A: Effects of MeOH on Protein Modificaiton ………………………….……………76

A.1 Background……………………......…………………………………………………77

A.2 Results and Discussion……………..………………………………………...………78

A.3 Experimental………………………...………………………………………..………80

Bibliography …………………………………………………………………………….………82

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GLOSSARY

APS………………………………………………………………………… Ammonium Persulfate

Ar………………………………………………………………………………………...Argon Gas

ATP .………………………………………………………………………Adenosine Triphosphate

ClpBQ……………………………………………………..2-Chlorocyclohexa-2,5-diene-1,4-dione

Cys…………………………………………………………………………………………Cysteine

Da ………………………………………………………...Dalton (equal to 1 amu; 1.66 x 10-27

kg)

DAB…………………………………………………………………………...1,2-Diacetylbenzene

DNP …………………………………………………………….……..2,4-Dinitrophenylhydrozine

kDa ……………………………...………………..Kilodalton (equal to 1000 amu; 1.66 x 10-24

kg)

Lys…………………………………………….……………………………………………..Lysine

NAD+

…………………………………………...Nicotinamide Adenine Dinucleotide (oxidized)

NADH ……………………………………………..Nicotinamide Adenine Dinucleotide (reduced)

MeOH………………………………………………...………………..Methanol (Methyl Alcohol)

PAH………………………………………………………...…..Polycyclic Aromatic Hydrocarbon

RNase A……………………………………………………...……………………..Ribonuclease A

SDS …………………………………………………………...…………..Sodium Dodecyl Sulfate

SDS-PAGE…………….…………..Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis

TCA…………………….……………………………………………………..Trichloroacetic Acid

TEMED………………………………………………..N,N,N’,N’-tetramethyl-ethane-1,2-diamide

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CHAPTER 1

INTRODUCTION

6

Before Van Leeuwenhoek developed the first microscope and observed the “animalcules”

now call bacteria, diseases were considered punishment from the divine (1-2). Today, science

has advanced at an exponential rate and continues to push the limits of the imagination. Despite

all the great accomplishments of the past centuries, there still exist many questions to be

answered within the universe. Thus it is critical to our existence to continue pushing the

scientific frontier and find answers to new problems.

One problem that would have never crossed Leeuwenhoek’s mind is the toxicity of

polycyclic aromatic hydrocarbons (PAHs). PAHs are known to exist in the environment. They

accumulate from various industrial processes like oil refinement and fossil fuel combustion (2-6)

and are even found in mainstream cigarette smoke (7). The most simple and well known PAH is

benzene. Among the scientific and medical community benzene has been suspected as a

carcinogen (4; 8-10), though the exact mechanism of toxicity is not well understood.

When introduced into a biological system, benzene can be metabolized into one of

several oxidized species such as a quinone or hydroquinone (4, 10-12). A proposed benzene

metabolism is shown in Figure 1.1. In the liver, the enzyme cytochrome P450 monooxygenase

generates an epoxide on the ring, which can then react through a variety of pathways. One

involves a spontaneous rearrangement generating a phenol. The phenol can then react with

cytochrome P450 monooxygenase again generating a second epoxide. This again rearranges

resulting in the formation of a hydroquinone. The hydroquinone is capable of redox cycling

which generates a quinone.

7

O

OH OH

O

OH

OH

O

O

A B C D E

Figure 1.1: Metabolism of benzene. Step A induced by cytochrome P450 monooxygenase; step

B nonenzymatic rearrangement; step C induced by cytochrome P450 monooxygenase; step D

nonenzymatic rearrangement; step E redox cycling.

Due to their reactivity, PAH quinones can modify biological molecules such as lipids,

nucleic acids, and proteins (8-9, 13-20). Modification of these biological molecules could be

detrimental to the health of a cell since many cellular components function based on their

structure. The mechanism of modification induced by the PAH quinones is not fully understood,

however it is critical to understand the extent of the modification to determine if the PAH

quinones present any threat to humans or the environment.

Because little work has been done to study how PAH quinones effect cellular proteins,

our laboratory is interested in investigating the modification of protein induced by the PAH

quinones, using Ribonuclease A (RNase A) and 2-chlorocyclohexa-2,5-diene-1,4-dione

(chlorobenzoquinone or ClpBQ) as our model system. ClpBQ was chosen as one of three

quinones to be studied by various projects in our laboratory. The other two are 2-

methylcyclohexa-2,5-diene-1,4-dione (methylbenzoquinone or MepBQ) and cyclohexa-2,5-

diene-1,4-dione (benzoquinone or pBQ). Together these three quinones represent a broad range

of simple PAH metabolites. These PAH metabolites are shown in Table 1.1 along with the

PAHs they come from. It should be noted that the metabolism of all of these PAHs is expected

to follow the same pathway as that of benzene.

Quinone

(oxidized form) Hydroquinone

(reduced form)

8

PAH PAH Metabolite

Unsubstituted

benzene

O

O Benzoquinone (BQ)

Electron Donating

Me

methylbenzene

O

O

Me

Methylbenzoquinone (MepBQ)

Electron Withdrawing

Cl

chlorobenzene

O

O

Cl

Chlorobenzoquinone (ClpBQ)

Table 1.1: The selected PAHs for the study in our laboratory and their respective metabolites.

This project only focuses on chlorobenzoquinone, shown in the grey.

To better understand the nature of the PAH quinone induced modification of cellular

protein, this project is focused on examining the effects of modification at both the chemical

level and the protein level.

Chemical Model System:

The primary objective of this series of experiments is reacting ClpBQ with select

nucleophilic amino acids to determine kinetic data. The experiments were carried out by

monitoring the change in a sample containing ClpBQ and the selected amino acid such as lysine

9

or cysteine, over a period of time. The time-dependent change in the spectral features can be

monitored using UV/Vis spectroscopy, and can be used to calculate kinetic data. More specific

details for these experiments are presented in the background information of Chapter 2.

Protein Model System:

The series of experiments were focused on investigating the modification of RNase A

induced by ClpBQ. After RNase A is treated with ClpBQ at various concentrations and time

periods, we observed protein modification using several laboratory techniques. Oxidative

damage can be detected by treating a modified protein sample with dinitrophenylhydrazine

(DNP). DNP can form a covalent bond to the oxidized sites on a protein. Because DNP

derivatives have characteristic UV/Vis spectral feature, we can determine if the target protein has

been modified by evaluating the UV/Vis data following a workup with DNP. In order to detect

protein crosslinking, we will carry out electrophoresis experiments. Using this technique the size

of the RNase A can be determined relative to a control RNase A. More specific details for these

experiments are presented in the background information of Chapter 3.

10

References:

1. Gribbin, John. The Scientists. (2002) New York, NY. Random House

2. Porter Roy. The Greatest Benefit to Mankind; A Medical History of Humanity. (1997)

New York, NY. W. W. Norton & Company.

3. Giger, W.; Blurner, M. Polycyclic aromatic hydrocarbons in the environment. Isolation

and characterization by chromatography, visible, ultraviolet, and mass spectrometry,

Anal. Chem. 1974, 46, 1663-1671.

4. Waidyanatha, S.; Sangaiah, R.; Rappaport, S.M. Characterization and quantification of

cysteinyl adducts of benzene diol epoxide, Chem Res Toxicol. 2005, 18, 1178-1185.

5. Benner, Bruce A Jr.; Brynre, Nelson P.; Wise, Stephen A.; Mulholland, George W.; Lao,

Robert C.; Fingas, Mervin F. Polycyclic aromatic hydrocarbon emissions from the

combustion of crude oil on water. Environ. Sci. Technol. 1990, 24, 1418-1427.

6. Garrett, R.; Guenette, C.; Haith, C.; Prince, R.; Pyrogenic Polycyclic Aromatic

Hydrocarbons in Oil Burn Residues, Environ. Sci. Technol. 2000, 34, 1934-1937.

7. Ding, Y.; Ashley, D.; Watson, C. Determination of 10 Carcinogenic Polycyclic Aromatic

Hydrocarbons in Mainstream Cigarette Smoke. J. Agric, Food Chem. 2007, 55, 5966-

5973.

8. Mason, D.; Liebler, D. Characterization of Benzoquinone—Peptide Adducts by

Electrospray Mass Spectrometry, Chem. Res. Toxicol. 2000, 13, 976-982.

9. Osborne, R.; Raner, G.; Hager, L.; Dawson, J. C. funago Chloroperoxidase is also a

Dehaloperoxidase: Oxidative Dehalogenation of Halophenols, J. Am. Chem. Soc. 2006,

128, 1036-1037.

10. Bolton, J.; Trush, M.; Penning, T.; Dyryhurst, G.; Monks, T. Role of Quinones in

Toxicology, Chem. Res. Toxicol. 2000, 13, 135-160.

11. McLean, M. R.; Bauer, U.; Amaro, A. R.; Robertson, L. W. Identification of catechol and

hydroquinone metabolites of 4-monochlorobiphenyl, Chem Res Toxicol 1996, 9, 158-

164.

12. Snyder, R.; Hedli, C. C. An Overview of Benzene Metabolism, Environ Health Perspet

1996, 104 (Suppl. 6), 1165-1172.

13. Chenna, A.; Hang, B.; Rydberg, B.; Kim, E.; Pongracz, K.; Bodell, W. J.; Singer, B. The

benzene metabolite p-benzoquinone forms adducts with DNA bases that are excised by a

repair activity from human cells that differs from an ethenoadenine glycosylase, Proc

Natl Acad Sci USA 1995, 92, 5890-5894.

11

14. Nakayama, A.; Kawanishi, M.; Takebe, H.; Morisawa, S.; Yagi, T. Molecular analysis of

mutations induced by a benzene metabolite, p-benzoquinone, in mouse cells using a novel

shuttle vector plasmid, Mutat Res 1999, 444, 123-131.

15. Fisher, A. A.; Labenski, M. T.; Malladi, S.; Gokhale, V.; Bowen, M. E.; Milleron, R. S.;

Bratton, S. B.; Monks, T. J.; Lau, S. S. Quinone electrophiles selectively adduct

“electrophile binding motifs” within cytochrome c, Biochemistry 2007, 46, 11090-11100.

16. McCracken, P.; Bolton, J.; Thatcher, G. Covalent Modificaiton of Proteins and Peptides

by the Qunone Methide from 2-tert Butyl-4,6-dimethylphenol: Selectivity and Reactivity

with Respect to Competitive Hydration, J. Org. Chem. 1997, 62, 1820-1825.

17. Wang, X.; Thomas, B.; Sachdeva, R.; Arterburn, L.; Frye, L.; Hatcher, P.; Cornwell, D.;

Ma, J. Mechanism of arylating quinone toxicity involving Michael adduct formation and

induction of endoplasmic reticulum stress, PNAS. 2006, 103, 3604-3609.

18. Kondrová, E.; Stopka, P.; and Souček, P. Cytochrome P450 destruction benzene

metabolites 1,4-benoquinone and 1,4-hydroquinone and the formation of hydroxyl

radicals in minipig liver microsomes, Toxicol in Vitro 2007, 21, 566-575.

19. Hanzlik, R. P.; Harriman, S. P.; Frauenhoff, M. M. Covalent binding of benzoquinone to

reduced ribonuclease. Adduct structures and stoichiometry, Chem Res Toxicol 1994, 7,

177-184.

20. Dong, S.; Fu, P.; Shirsat, R.; Hwang, H.; Leszczynski, J.; Yu, H. UVA Light-Inducted

DNA Cleavage by Isomeric Methylbenz[a]anthracenes, Chem. Res. Toxicol. 2002, 15,

400-407.

12

CHAPTER 2

CHEMICAL MODEL REACTIONS

13

2.1 BACKGROUND

We sought to examine the reactions of ClpBQ with various amino acids. The two amino

acids we chose were lysine and cysteine. Their structures at pH 7.0 are shown below (1):

+H3N

O-

O

R

SH

NH3+Lysine; R =

Cysteine; R =

General form of amino acid

Both lysine and cysteine have lone pair electrons on their side chains. Because of these

electrons, these amino acids may be able to nucleophillicly attack an electron-deficient carbonyl

carbon. In particular, cysteine has been shown to react with PAH metabolites (2).

It is not the purpose of this experiment to determine exactly what type of reaction

occurred, but rather to determine if a reaction occurs and if so, how quickly. Thus the following

experiment focuses on the kinetics of such a reaction. It is the specific goal of these experiments

to calculate the rate constants for the reactions of ClpBQ with lysine cysteine respectively.

Below is a diagram showing the progress of the reaction:

Quinone Intermediate Product

The quinone first will react with an amino acid forming an intermediate species. The

intermediate may continue to react until a final product is formed. Each step of this reaction will

have a specific rate constant (k). This laboratory is interested in the reaction of the quinone with

an amino acid, therefore from the schematic above, we seek to calculate k1.

Amino Acid

k1 k2

14

The rate of reaction is proportional to the concentrations of the reactions raised to the nth

power, as shown in Equation 2.1. This is the general rate law (3).

R = k[A]a[B]

b Equation 2.1

R = Rate

k = Rate Constant

[A] = Concentration of Species “A”

a = Order of reaction with respect to species “A”

[B] = Concentration of Species “B”

b = Order of reaction with respect to species “B”

It is very difficult to elucidate a rate constant from this relationship where both [A] and

[B] are changing, thus this we sought a simpler solution. In order to determine k, this laboratory

will utilize pseudo first order kinetics. This is a technique that involves making a reaction

mixture such that one reactant is in great excess relative to the other. This in effect removes one

of the reactants from Equation 2.1 leading to Equation 2.2.

R = k[A] Equation 2.2

The effect of reactant B can be ignored because there will be little observed change in its

concentration compared to reactant A. In other words, all of reactant A is used before any

measurable amount of reactant B is consumed.

Beer’s Law (Equation 2.3) shows the direction relationship between concentration and

absorbance (4).

A = εbc Equation 2.3

A = Absorbance

ε = Extinction Coefficient

b = Path Length

c = Concentration

15

Using this relationship, it is possible to monitor the change in concentration of a reactant

using UV/Vis spectroscopy. By monitoring a specific chromophore as the reaction progresses, it

is possible to generate data that can be used to calculate the rate constant.

In order to know what wavelength(s) can be monitored and at what concentrations

UV/Vis spectroscopy can be used to detect the reactant, a standard curve must be generated.

This is accomplished by preparing a series of solutions containing known concentrations of

ClpBQ, irradiating them with the entire UV/Vis spectrum and determining where the relative

λmax are. The end result will show at what wavelengths ClpBQ absorbs UV/Vis radiation and

what range of concentrations can be detected by this laboratory’s instrumentation.

From the rate law for first order kinetics, comes Equation 2.4, which shows the

relationship between absorbance and the rate constant (5). This is the equation used to calculate

the rate constant.

ln �� − �� − �

= −�

��= Absorbance at infinity

�� = Absorbance at a given time

�� = Initial Absorbance

k = Rate Constant

t = Time

In order to use the above equation, an absorbance at infinity needed to be determined.

This laboratory decided to use 8 times the half-life (8t1/2) of this reaction as the absorbance at

infinity. 8t1/2 was chosen via methodology development; there is very little difference in

absorbance at 8t1/2 of a reaction and time periods beyond.

To determine the half-life, a plot of absorbance verses time is generated in the linear

region of the graph. Using Excel, a curve was generated to fit to the data. This curve is in the

general form of Equation 2.5.

Equation 2.4

16

� = �� + � Equation 2.5

y = Absorbance

m = Slope (a function of the rate constant)

x = Time

b = Initial Concentration of Reactant

The half-life is calculated by rearranging equation 2.5 and dividing by 2 which yields

Equation 2.6.

− �� = �

� Equation 2.6

�� = half-life

Once the absorbance at 8t1/2 is observed, the rate constant for a given reaction can be

calculated using equation 2.4.

17

References:

1. Boyer, R. Concepts in Biochemistry; Third Edition. Hoboken, NJ. John Wiley & Sons, Inc.,

2006.

2. Waidyanatha, S.; Sangaiah, R.; Rappaport, S. M. Characterization and Quantification of

Cysteinyl Adducts of Benzene Diol Epoxide. Chem. Res. Toxicol, 2005, 18, 1178-1185.

3. Brady, J. E.; Senese, F. Chemistry Matter and Its Changes; Fourth Edition. Hoboken, NJ.

John Wiley & Sons Inc., 2004.

4. Skoog, D. A.; Holler, F. J.; Crouch, S. R. Principles of Instrumental Analysis; Seventh Edition.

Belmont, CA. Thomson Higher Education, 2007.

5. Atkins, P.; Paula, J. Atkins' Physical Chemistry; Eighth Edition. New York, NY. W. H.

Freeman and Company, 2006.

18

2.2 RESULTS AND DISCUSSION

2.2.1 Standardization Curve for ClpBQ

The standard curve for ClpBQ at various concentrations is shown in Figure 2.1. In the

curve (200-700 nm), two λmax were identified, one at 255 nm and the other at 330 nm.

The plot of absorbance at 255 nm versus the concentration of ClpBQ (Figure 2.2) shows

both a region where the absorbance increases linearly, and a region where absorbance is too great

and the detector can no longer generate linear data. A plot of 0.01 to 0.15 mM ClpBQ verses

time is shown in Figure 2.3. According to this figure, the absorbance at 255 nm increases

linearly from 0.01 to 0.15 mM. At concentrations higher than 0.15 mM, the solution absorbs too

much at 255 nm and the curve is no longer linear. Thus the best range to observe the

concentration of ClpBQ at 255 nm is between 0.01 and 0.15 mM.

The plot of absorbance at 330 nm versus concentration is shown in Figure 2.4. The

entire range increases linearly for all concentrations of ClpBQ examined in this experiment.

Thus this wavelength may be used to monitor ClpBQ from 0.010 to 0.325 mM.

2.2.2 Kinetics of ClpBQ with Lysine

For the kinetic analysis, the absorbance change at both λ255 and λ330 were utilized. A

pseudo-first order condition was used where the concentration of ClpBQ (0.10 mM) was much

lower than that of lysine (20.0 mM). Thus there should only be an observable change in the

concentration of ClpBQ and no observable change in the concentration of lysine.

Figure 2.5 shows a representative UV/Vis spectrum of the reaction between ClpBQ and

lysine, with a scan occurring every 35 sec. At first glance it becomes evident that a reaction did

19

occur because the spectrum changes over time. The λmax at 255 nm slowly disappears while the

chromophore at 330 nm is covered up by an adjacent spectral feature which develops over time.

Thus in this reaction, only the absorbance at 255 nm can be used for calculating the rate constant.

In addition, it should be noted, two spectral features of interest occur at 290 and 515 nm. At

these wavelengths the absorbance of the reaction increases and then decreases suggesting a

chemical species formed and then was consumed in another reaction, indicative of an

intermediate.

Figure 2.6 is the average change in absorbance at 255 nm over the 35 min reaction time

period. From this spectrum, it becomes evident that a reaction is occurring where ClpBQ is

consumed due to the linear decrease in absorbance (and thus concentration; see Equation 2.3) at

255 nm.

In order to calculate the rate constant, the absorbance at infinity must be determined. Our

laboratory used 8t1/2 as absorbance at infinity. To calculate the half-life, the absorbance of the

reaction of ClpBQ with lysine was plotted versus time for the first 4 min (Figure 2.7). This

spectrum shows a region where the concentration of ClpBQ is decreasing linearly. Microsoft

Excel was used to generate a curve to fit these data. Using Equation 2.6, the half-life was

calculated to be about 4.1 min. Thus the absorbance at infinity (8t1/2) will occur at 34 min. The

reaction cuvets were left in the temperature chamber of the UV/Vis spectrophotometer until a

scan could be taken about 34 min, and thus an absorbance measurement obtained for absorbance

at infinity.

Based on the Equation 2.4, the line generated by plotting ln ��

� versus time will

have a negative slope equal to the rate constant. Figure 2.8 is a resulting curve from this plot. 2

similar plots were generated by repeating this reaction. The rate constant from each was

20

calculated and averaged. The rate constant for the reaction of ClpBQ (0.100 mM) and Lys (20.0

mM) is 0.2258 min-1

± 0.02133.

2.2.3 Kinetics of ClpBQ with Cysteine

This kinetic study was carried out in a similar fashion to the kinetic experiment with

lysine, however the results could not yield kinetic data. Figure 2.9 shows the UV/Vis spectra

generated by scanning every 30 min for 5 hours. At both 255 nm and at 330 nm there is no

significant decrease in absorbance over the time period observed. Based on this observation it

appears that the reaction of ClpBQ with cysteine is slow.

To further study this reaction, the reaction was incubated for 3 days at 37 °C, and daily

scans were examined. Figure 2.10 shows how the absorbance profile changed over time.

Because there is no observable decrease in absorbance at 255 nm or 330 nm, kinetic data cannot

be determined from this data by examining a changing concentration of ClpBQ. However

because the spectra does change over time, a reaction is occurring. To determine the nature of

this reaction will require further investigation by other methods.

21

2.3 CONCLUSIONS FROM CHEMICAL MODEL REACTIONS

In conclusion, lysine reacts with ClpBQ. Pseudo first order kinetic conditions and

UV/Vis spectroscopy were used to calculate the rate constant for the reaction of lysine with

ClpBQ. By monitoring the steady decrease in absorbance at 255 nm, the rate constant for the

reaction of ClpBQ (0.100 mM) and Lys (20.0 mM) at 37 °C is 0.2258 min-1

± 0.02133.

The rate constant for the reaction between cysteine and ClpBQ could not be calculated

using Pseudo first order kinetics and UV/Vis spectroscopy. The two λmax used to monitor the

concentration of ClpBQ were covered by other spectral features throughout the duration of the

reaction. However, because spectral data changes over a three day incubation period, it is with

certainty we conclude a reaction does occur between cysteine and ClpBQ, though to understand

the exact nature of this reaction will require further study.

22

Figure 2.1: UV/Vis spectra of ClpBQ in phosphate buffer (pH 7.0; 50.0 mM) at 37 °C (0.010,

0.025, 0.050, 0.075, 0.100, 0.125, 0.150, 0.175, 0.200, 0.225, 0.250, 0.275, 0.300, and 0.325

mM) using a 1.0 cm path length quartz cuvet (data duplicated).

0

0.5

1

1.5

2

2.5

3

200 250 300 350 400 450 500 550 600 650 700

Absorbance

Wavelength (nm)

ClpBQ spectrum; Changing Concentration

Figure 2.2: Plot of absorbance at 255 nm versus concentration of Cl

buffer (pH 7.0; 50 mM) at 37 °C using a 1.0

0

0.5

1

1.5

2

2.5

3

0 0.05 0.1

Absorbance

Plot of absorbance at 255 nm versus concentration of ClpBQ (mM) in phosphate

at 37 °C using a 1.0 cm path length quartz cuvet (data duplicated)

0.1 0.15 0.2 0.25

Concentration (mM)

ClpBQ at 255 nm

23

BQ (mM) in phosphate

cm path length quartz cuvet (data duplicated)

0.3 0.35

Figure 2.3: Linear range of absorbance at 255 nm versus concentration of Cl

phosphate buffer (pH 7.0; 50 mM)

duplicated)

0

0.5

1

1.5

2

2.5

3

0 0.02 0.04

Absorbance

Linear range of absorbance at 255 nm versus concentration of ClpBQ (mM) in

phosphate buffer (pH 7.0; 50 mM) at 37 °C using a 1.0 cm path length quartz cuvet (data

y = 15.424x + 0.0679

R2 = 0.9973

0.06 0.08 0.1 0.12

Concentration (mM)

ClpBQ at 255 nm

24

BQ (mM) in

at 37 °C using a 1.0 cm path length quartz cuvet (data

0.14 0.16

Figure 2.4: Plot of absorbance at 330 nm versus concentration of Cl

buffer (pH 7.0; 50 mM) at 37 °C using a 1.0 cm path length quartz cuvet (data duplicated)

0

0.05

0.1

0.15

0.2

0.25

0 0.05

Absorbance

Plot of absorbance at 330 nm versus concentration of ClpBQ (mM) in phosphate


y = 0.6551x + 0.0038

R2 = 0.9986

0.1 0.15 0.2 0.25

Concentration (mM)

ClpBQ at 330 nm

25

BQ (mM) in phosphate


0.3 0.35

26

Figure 2.5: Representative UV/Vis spectra for the reaction of ClpBQ (0.1 mM) with lysine (20.0

mM) in phosphate buffer (pH 7.0; 50 mM) at 37 °C using a 1.0 cm path length quartz cuvet (data

triplicated). Arrows indicate direction absorbance changes as a function of time; side-by-side

arrows indicate an increase followed by a decrease.

0

0.5

1

1.5

2

2.5

200 250 300 350 400 450 500 550 600 650 700

Absorbance

Wavelength (nm)

0.1 mM ClpBQ; 20 mM Lys

27

Figure 2.6: Average absorbance change at 255 nm versus time for the reaction of ClpBQ (0.1

mM) and lysine (20.0 mM) in phosphate buffer (pH 7.0; 50 mM) at 37 °C using a 1.0 cm path

length quartz cuvet (data triplicated).

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

0 5 10 15 20 25 30 35 40

Absorbance

Time (min)

0.10 mM ClpBQ; 20.0 mM Lys (255 nm)

28

Figure 2.7: Linear range of the absorbance at 255 nm verus time for the reaction of ClpBQ (0.1

mM) and lysine (20.0 mM) in phosphate buffer (pH 7.0; 50 mM) at 37 °C using a 1.0 cm path

length quartz cuvet (data triplicated).

y = -0.213x + 1.735

R² = 0.995

0.6

0.8

1

1.2

1.4

1.6

1.8

2

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5

Absorbance

Time (min)

0.10 mM ClpBQ; 20 mM Lysine (255 nm)

29

Figure 2.8: Plot to determine the rate constant. Absorbance data generated from the reaction of

ClpBQ (0.10 mM) with lysine (20.0 mM).

y = -0.250x + 0.115

R² = 0.991

-1

-0.9

-0.8

-0.7

-0.6

-0.5

-0.4

-0.3

-0.2

-0.1

0

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5

Time (min)

Plot to Determine Rate Constant !

�" ∞−

" #" ∞

− " $

30

Figure 2.9: Representative UV/Vis spectra for the reaction of ClpBQ (0.1 mM) with cysteine

(20.0 mM) in phosphate buffer (pH 7.0; 50 mM) at 37 °C using a 1.0 cm path length quartz cuvet

(data triplicated). Arrows indicate absorbance change as a function of time.

0

0.5

1

1.5

2

2.5

3

200 250 300 350 400 450 500 550 600 650 700

Absorbance

Wavelength (nm)

0.10 mM ClpBQ; 20.0 mM Cys

31

Figure 2.10: Representative UV/Vis spectra for the reaction of ClpBQ (0.1 mM) with cysteine

(20.0 mM) in phosphate buffer (pH 7.0; 50 mM) at 37 °C using a 1.0 cm path length quartz cuvet

(data triplicated). Approximately three days of incubation time. Arrows indicate absorbance

change as a function of time.

0

0.5

1

1.5

2

2.5

3

200 300 400 500 600 700

Absorbance

Wavelength (nm)

0.10 mM ClpBQ; 20.0 mM Cys

Initial

21 Hours

45 Hours

69 Hours

32

2.4 EXPERIMENTAL

General:

UV/Vis spectra were obtained using Biospec-1601 spectrophotometer by Shimadzu with

a jacketed (temperature controlled) cell compartment to maintain physiological temperature.

Software used for data collection was UVProbe 2.30 by Shimadzu. Specifically using the “scan”

feature was utilized for kinetic study. Data analysis software employed was Excel 2007 by

Microsoft.

Water used in these experiments is always MilliQ water. Solvents and reagents used are

highest grade available from commercial sources.

2.4.1 Standard Curve for ClpBQ

A standard curve was constructed by plotting the absorbance at λmax and the various

concentrations of ClpBQ using UV/Vis spectroscopy. The final concentrations of ClpBQ used

for study are: 0.010, 0.025, 0.050, 0.075, 0.100, 0.125, 0.150, 0.175, 0.200, 0.225, 0.250, 0.275,

0.300, and 0.325 mM.

The diluted solutions were made by diluting a ClpBQ stock solution (14.03 mM) with 50

mM phosphate buffer (pH 7.0; 50 mM) as shown by Table 2.1. The stock solution of ClpBQ

was prepared by dissolving 4.0 mg of ClpBQ in 2.0 mL of 50 mM phosphate buffer (pH 7.0; 50

mM). The solution was sonicated for 20 min to dissolve ClpBQ. The stock solution was kept on

ice for the entire experiment.

33

Quartz cuvets were filled with the appropriate amount of phosphate buffer to achieve

each diluted solution to be detected with UV/Vis spectroscopy. Then, the calculated aliquots of

the cold ClpBQ stock solution were added to cuvets prior to each scan.

2.4.2 Kinetics of ClpBQ with Lysine

To 1.0 mL quartz cuvets was added 400 µL of lys (50.0 mM) in phosphate buffer (pH

7.0; 50 mM) and 593 µL of phosphate buffer (pH 7.0; 50 mM). Fresh ClpBQ stock solution

(14.03 mM) was prepared and kept on ice until the UV/Vis spectrophotometer was ready for use.

The solution in a cuvet was equilibrated to 37 ºC at least 5 min prior to initializing a reaction in

the temperature controlled cell holder on the UV/Vis spectrophotometer. 7.13 µL of ClpBQ

stock solution (14.03 mM) was added to the equilibrated samples. This made the final

concentration in the reaction cuvet 0.100 mM ClpBQ and 20.0 mM lysine. Then, the solution

was mixed by inversion and placed into the instrument for detection every 35 sec for 20 min.

2.4.3 Kinetics of ClpBQ with Cysteine

To quartz cuvets (1.0 mL) was added 400 µL of Cys (50.0 mM) in phosphate buffer (pH

7.0; 50 mM) and 593 µL of phosphate buffer (pH 7.0; 50 mM). Fresh ClpBQ stock solution

(14.03 mM) was prepared and kept on ice until the UV/Vis spectrophotometer was ready for use.

The solution in a cuvet was equilibrated to 37 ºC at least 5 min prior to initializing a reaction in

the temperature controlled cell holder on the UV/Vis spectrophotometer. 7.13 µL of ClpBQ

stock solution (14.03 mM) was added to the equilibrated samples. This made the final

concentration in the reaction cuvet 0.100 mM ClpBQ and 20.0 mM cysteine. Then, the solution

was mixed by inversion and placed into the instrument for detection every 30 min for 5 hr.

34

Table 2.1 Compositions of samples for ClpBQ standard curve

Note: to maximize efficiency, both 3.0 mL and 1.0 mL cuvets were utilized.

Final Concentration

0.010 mM 0.025 mM 0.050 mM 0.075 mM 0.100 mM 0.125 mM 0.150 mM

14.03 mM ClpBQ stock 2.14 µL 5.35 µL 10.69 µL 16.04 µL 7.13 µL 26.73 µL 10.69 µL

Phosphate Buffer (pH 7.0; 50 mM) 2.997 mL 2.994 mL 2.989 mL 2.984 mL 0.993 mL 2.973 mL 0.989 mL

Final Volume (mL) 3.0 mL 3.0 mL 3.0 mL 3.0 mL 1.0 mL 3.0 mL 1.0 mL

Final Concentration

0.175 mM 0.200 mM 0.225 mM 0.250 mM 0.275 mM 0.300 mM 0.325 mM

14.03 mM ClpBQ stock 37.42 µL 14.26 µL 16.04 µL 17.46 µL 19.6 µL 21.38 µL 23.16 µL

Phosphate Buffer (pH 7.0; 50 mM) 2.963 mL 0.986 mL 0.984 mL 0.982 mL 0.980 mL 0.979 mL 0.977 mL

Final Volume (mL) 3.0 mL 1.0 mL 1.0 mL 1.0 mL 1.0 mL 1.0 mL 1.0 mL

35

CHAPTER 3

PROTEIN MODEL REACTIONS

36

3.1 BACKGROUND

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is used by our

laboratory to analyze modified protein and is a common laboratory technique (1). SDS-PAGE

works by first breaking a protein into its primary structure and applying a net negative charge to

the entire molecule. To do this, a protein is heated in a solution containing 2-mercaptoethanol

and sodium dodecyl sulfate (SDS). The structure of molecules are shown below:

O S O-

O

O

Na+

SHHO

The combination of heat and 2-mercaptoethanol breaks all the disulfide bonds which are

reduced to thiol groups (R—SH). Then the free sulfur will bind to the sulfur of 2-

mercaptoethanol. Proteins in a natural confirmation depend on these disulfide bonds as a key

component which maintains their secondary and tertiary structure. Breaking these bonds results

in the protein unfolding and forming a linear structure. The other key component to preparing a

protein for SDS-PAGE involves applying a charge. SDS has a negative charge on its polar head

and also contains a non-polar tail. It can interact via electrostatic interactions such as hydrogen

bonding or van der Waals forces with both polar and non-polar residues on a protein. This coats

the entire protein, both the polar sections and the non-polar sections, giving the protein an overall

negative charge.

At this point, the negatively charged protein is loaded into a polyacrylamide gel. The

polyacrylamide gel is a matrix which offers resistance to the protein as it travels; think of the gel

as containing many holes and filled random spaces like Swiss cheese. Once the protein is loaded

into the top of the gel, an electric current is sent through wires at the top and bottom of the gel.

2-mercaptoethanol sodium dodecyl sulfate (SDS)

Na+

37

The wires are configured in a manner which generates a positive field at the bottom of the gel.

Thus the negatively charged protein will migrate towards the bottom of the gel once an electric

potential is applied. Once the voltage is removed, the distance the protein traveled through the

gel, is proportional to the size of the protein—a larger protein will encounter more resistance

from the gel than a smaller protein, and thus will not be able to travel as far in a given amount of

time. This separates proteins based on size.

It is common practice to run proteins of known size (molecular marker) alongside the

proteins of interest. By looking across the gel and comparing the known proteins to the

unknown proteins, a specific molecular weight of each protein can be determined.

ClpBQ is an oxidized compound with two carbonyl carbons. Because of these

characteristics there may be a variety of reactions it can induce. Our laboratory postulated three

possible modification pathways for the reaction of ClpBQ and a protein.

O

O

ClCl

OH

OH

Oxidation

Reduction

ProteinNu

LysH2N Protein

LysO Protein

+LysH2N Protein

Pathway I

Pathway II

Pathway IIIO2

O2-

ClpBQClpHQ

Adduct Formation

Protein Crosslinking

Oxidative Damage

38

Pathway I shows a nucleophilic residue attacking the reactive positions on ClpBQ. Many

amino acids can react as nucleophiles; lysine, serine, threonine, and cysteine have lone pair

electrons (1) and can act as a nucleophile attacking a carbonyl carbon or a double bond in

ClpBQ. The result of this type of reaction is adduct formation. Where on the protein this adduct

is formed will determine the effects on the protein’s structure and ability to perform its biological

function. If the adduct is created in the active site of an enzyme, it is unlikely the enzyme could

still catalyze its intended reaction.

Pathway II shows lysine oxidized to oxolysine. Lysine contains an epsilon amino group

which can be oxidized resulting in the formation of an aldehyde (2). This aldehyde can then

react with another lysine forming a covalent bond between the two amino acids. The specific

mechanism is still a subject of debate, but the end result could be crosslinked proteins. This

occurs when a lysine residue on protein A is oxidized and reacts with another lysine residue on

protein B resulting in a covalent bond between the two proteins. Because proteins function

based on their structure, this modification may cause the protein to not perform as nature

intended.

The redox cycling between the reduced and oxidized quinone can generate a superoxide

anion (O2-). This radical is a highly reactive species and will react with nearly anything it

contacts. There are many reactions which may occur with this molecule in a biological system,

and most likely the resulting reaction would damage cellular components. Our laboratory is not

specifically looking for this type of modification, but it is important to understand it is a

possibility.

39

1,2-Diacetylbenzene (DAB) is known to induce protein crosslinking (3). In order to fully

understand the type of modification induced by ClpBQ, our laboratory compared the type of

modification induced by DAB as a control with the modification induced by ClpBQ. In other

words, RNase A will be modified by DAB to serve as a control experiment. The structure of

DAB is shown below:

O

O

The specific aims of the project are i) to determine if ClpBQ can cause protein

crosslinking and adduct formation. To study this, we have selected RNase A to be the model

protein. SDS-PAGE was used to determine if these types of modification are induced by ClpBQ.

And ii) to examine the possible incubation pathway for the ClpBQ induced protein modification.

Since it has been proposed that ClpBQ modifies protein through oxidation pathways, we

attempted to examine the effects of reducing agents on the modification induced by ClpBQ on a

model protein. The choice of reducing agents includes nicotinamide adenine dinucleotide

(NADH) and L-ascorbic acid (vitamin C). Both of these molecules are commonly found in

biological systems (1). NADH is critical to the cell with regard to energy production. During

cellular metabolism NADH (the reduced form) is generated through a variety of pathways and,

following its formation, it is oxidized to NAD+ in the electron transport chain. This process

generates adenosine triphosphate (ATP), the molecule used for energy in cellular processes. The

structure of NADH is shown below (1):

1,2-diacetylbenzene (DAB)

40

N

O

H2N

O

HO

HO

O

P O-

O

OP

O

O

-O

O

HO

HON N

N

N

NH2

Animals must consume L-ascorbic acid in their diets because it is not synthesized in

animal cells; however it is essential to life. Most notably L-ascorbic acid is an antioxidant; it

reacts with reactive oxygen species which could cause cell damage. This process oxidizes L-

ascorbic acid which is outlined below (1):

O

OHO

HO

HO

OH

O

OO

O

OH

OH

Oxidation

Reduction

nicotinamide adenine dinucleotide (NADH)

L-ascorbic acid

reduced

L-ascorbic acid

oxidized

41

Since both NADH and L-ascorbic acid can reduce other molecules, this lab seeks to

determine if modification of protein caused by ClpBQ and be inhibited by NADH and L-ascorbic

acid.

In addition to determining the type of modification induced by ClpBQ, we were

interested in determining if lysine is oxidized in RNase A. When lysine is oxidized the epsilon

amino group is replaced by an aldehyde. Natural proteins contain no ketones or aldehydes. Thus

if we detect an aldehyde in the protein, then it is confirmed that lysine has been oxidized. To

detect aldehydes we use 2,4-Dinitrophenylhydrazine (DNP). DNP will covalently bond with

carbonyl groups in the presence of acid to form an imide (4). Then, the complex of DNP and

the trapped carbonyl compound is detected by UV/Vis spectroscopy from 350 nm to 450 nm by a

unique spectral feature (5). Thus if DNP binds to a carbonyl group in modified RNase A, a

specific spectral feature will appear in the UV/Vis spectrum of modified RNase A. The structure

of DNP along with a summary of this reaction is shown below:

Lys NH2

O

O

Cl

Lys ODNP

Modified Lys DNP

(Oxolysine)

NO2

NO2

NHH2N

2,4-Dinitrophenylhydrazine (DNP)

42

References:

1. Boyer, R. Concepts in Biochemistry; Third Edition. (2006) Hoboken, NJ. John Wiley & Sons.

Inc.

2. Eilstein J., Giménez-Arnau, E., Duché, D., Rousset, Françoise. Mechanistic Studies on the

Lysine-Induced N-Formylation of 2,5-Dimethyl-p-benzoquinonediimine, Chem. Res. Toxicol

2007, 20, 1155-1161.

3. Kim, M. S., Hashemi, S. B., Spencer, P. S., and Sabri, M. I. (2002) Amino acid and protein

targets of 1,2-diacetylbenzene, a potent aromatic gamma-diketone that induces proximal

neurofilamentous axonopathy, Toxicol Appl Pharmacol 183, 55-65

4. Bruice, P. Y., Organic Chemistry; Fifth Edition. (2007) Upper Saddle River, NJ. Pearson

Prentice Hall.

5. Levine, R. L.; Williams, J. A.; Stadtman, E. R.; Shacter, E. Carbonyl assays for determination

of oxidatively modified proteins, Methods Enzymol 1994, 233, 346-357

43

3.2 RESULTS AND DISCUSSIONS

3.2.1 ClpBQ Concentration Dependent Modification of RNase A

Unmodified RNase A has a molecular weight of 14 kDa, and a solution containing

unmodified RNase A is colorless. Unmodified RNase A is shown in lane 1 and 2 (L1 and L2) of

Figure 3.1. Moving from left to right across this gel, the concentration of ClpBQ increases

while all other conditions remain the same. The wells on the right side of this gel contain bands

of greater molecular weight which are not present in the control; this suggests RNase A was

modified by ClpBQ in a concentration dependent manner. To further support this idea, it was

observed that solutions containing RNase A and ClpBQ appeared to be brown after incubation,

while solutions containing only RNase A remained colorless after incubation. Based on these

observations, it is clear that RNase A is modified by ClpBQ. Specifically, the SDS-PAGE data

suggest that the modified RNase A, with a higher molecular weight, could be induced by adduct

formation or crosslinking.

Upon further analysis of Figure 3.1, it appears that as the concentration of ClpBQ in a

solution increases, RNase A is modified to a greater extent. Lane 3 (L3) contained 0.1 mM

ClpBQ and shows minimal modification of RNase A while lane 4 (L4) contained 0.5 mM ClpBQ

(5-fold increase from lane 3) and shows a clear well-developed second band. Furthermore lane

6 (L6) contained 5.0 mM ClpBQ (50-fold increase from lane 3), and a series of bands with

various molecular weights evolved in this lane. This fully supports the idea that modification

induced by ClpBQ occurred to a greater extent at higher concentration.

44

3.2.2 1,2-Diacetylbenzene Dependent Modification of RNase A

Figure 3.2 shows the SDS-PAGE data from 1,2-diacetylbenzene (DAB) modification of

RNase A. Lane 1 (L1) shows unmodified RNase A while the remaining lanes contain reaction

mixtures of DAB (1.0 mM) with RNase A after incubation. It becomes clear upon comparing

these lanes that DAB modifies RNase A similar to the modification induced by ClpBQ. The

bands of greater molecular weight resulting from DAB modification are in similar positions

when compared to the bands of greater molecular weight resulting from ClpBQ modification.

Since DAB is known to induce protein crosslinking, the similar data generated by both DAB and

ClpBQ suggests ClpBQ can induce protein crosslinking as well.

However there is a key difference between modification induced by DAB and

modification induce by ClpBQ. Following the incubation of RNase A with DAB, the solution

appeared purple. Recall the ClpBQ induced a brown color in the incubated solution. The color

difference in modified RNase A induced by DAB and ClpBQ, respectively, strongly suggests

that the nature of the protein modification involves not only crosslinking but also adduct

formation with each reagent.

3.2.3 ClpBQ Time and Concentration Dependent Modificaiton of RNase A

A series of reactions were carried out to examine the time and concentration dependent

effects on the modification of RNase A by ClpBQ. RNase A was incubated at 3 different

concentrations of ClpBQ (0.5, 1.0 and 5.0 mM). Incubation time intervals ranged from 10 min to

5 hr. All the results of this experiment show RNase A was modified by ClpBQ.

Figure 3.3(A/B) represents RNase A upon incubation with 5.0 mM ClpBQ at various

time points and the modification was visualized by SDS-PAGE. Moving left to right across

45

these gels, incubation time increases. Accordingly, the intensity of modification increases

moving left to right across these gels. This observation becomes most clear by comparing lane 2

(L2) and lane 6 (L6) in Figure 3.3A. L2 represents the reaction which was incubated for 10 min

while L6 represents the reaction which was incubated for 50 min. The top of L6 shows a dark

blue band indicative of a large protein, most likely highly crosslinked. The same position of L2

contains no band. This observation (seeing how all other lanes show a general trend towards

greater modification with longer incubation) suggests a longer incubation period with ClpBQ

causes more modification of RNase A.

All the lanes except for the control in lane 1 (L1) in Figure 3.3B appear to be similar and

all show modification of RNase A. When comparing lane 2 (L2), which was incubated for 1 hr,

to lane 6 (L6), which was incubated for 5 hr, it is obvious there is very little difference between

them. This finding suggests that the modification of RNase A by 5.0 mM ClpBQ completes in 1

hr and thus there is no more noticeable progress to be observed passing 1 hr incubation time.

The gels of Figure 3.4(A/B) show the incubation time effects on the modification of

RNase A induced by 1.0 mM ClpBQ. These results show a similar trend to that of the incubation

reaction with 5.0 mM ClpBQ. As the incubation time increases, so does the extent of protein

modification. In Figure 3.4A comparing lane 2 (L2), which represents the effects of a 10 min

incubation, to lane 5 (L5), which reflects the effects of a 40 min incubation, shows the clear

development of a third band with a molecular weight around 60 kDa. L2 shows no band in this

region, while in L5 it is clearly visible.

Figure 3.4B represents the incubation reactions where RNase A was incubated with 1.0

mM ClpBQ for 1, 2, 3, 4, and 5 hr, respectively. Careful examination of lane 3 (L3) through

lane 6 (L6) shows no significant difference among the observed modification. L3 correlates to a

46

2 hr incubation period and L6 shows the modified RNase A after a 5 hr incubation time.

Interestingly, lane 2 (L2), which shows RNase A modified with 1.0 mM ClpBQ for 1 hr, shows

different features from the other lanes. Comparing the top of L2 to top of L3 reveals an obvious

smear from the top of the gel to the first discrete band only present in L3 and missing from L2.

This smear is consistent in all other lanes which suggest at 1.0 mM concentration of ClpBQ,

modification of RNase A is completed after 2 hr rather than 1 hr with 5.0 mM ClpBQ.

The SDS-PAGE data of 0.5 mM ClpBQ modified RNase A are shown in Figure

3.5(A/B). Figure 3.5A shows the modification of RNase A with 0.5 mM ClpBQ for 10, 20, 30,

40 and 50 min respectively. Except the control lane (L1), all lanes look remarkably similar at

first glance. Only upon careful examination does one see a difference among the lanes.

Comparing lane 2 (L2) to lane 6 (L6) it becomes obvious. The space between the wells is the

color of the gel without protein in it. When comparing the upper half of L6 to this space, it

becomes clearer that the protein in L6 is more modified than that in L2. The upper half of L2 is

the same color as the space adjacent to it, thus there is not any protein present.

Figure 3.5B shows the same reaction mixture, but with incubation times at 1, 2, 3, 4 and

5 hr, respectively. There does not appear to be any obvious difference among the bands in the

gel representing modified RNase A. It is clear, however, that RNase A is modified by ClpBQ

and the modification becomes more extensive with longer incubation time. However, further

modification did not appear to occur to a greater extent with longer incubation times as it did

with 5.0 and 1.0 mM ClpBQ.

47

3.2.4 ClpBQ Modification of RNase A Inhibited by Concentration Dependent NADH

The inhibitory effects of NADH on the modification of RNase A by ClpBQ were

examined and the results are shown in Figure 3.6. Moving from left to right across this gel, the

concentration of ClpBQ is kept constant, while the concentration of NADH increases. As shown

in Figure 3.6, it is apparent that increasing the concentration of NADH inhibits protein

modification. Lane 2 (L2) shows RNase A modified by ClpBQ (5.0 mM) with no NADH

present. Moving to the right the extent of modification appears to be suppressed. Lane 4 (L4)

contains an equal concentration of NADH and ClpBQ and protein modification is still observed

in this condition. Lanes 5 and 6 (L5 and L6) contain excess NADH and show modification

further inhibited. Lane 6 represents a reaction mixture that contained 50 mM NADH (10-fold

higher concentration of ClpBQ) and shows almost no modification of RNase A. It appears

through some mechanism, NADH can inhibit modification of RNase A induced by ClpBQ.

3.2.5 ClpBQ Modification of RNase A Inhibited by Concentration Dependent L-Ascorbic

Acid

The inhibitory effects of L-ascorbic acid on the modification of RNase A by ClpBQ were

evaluated. Figure 3.7 is a photograph of the microcentrifuge tubes which contained the reaction

at various conditions. Moving from left to right across the photograph, the concentration of L-

ascorbic acid in solution increases while the concentration of ClpBQ is kept constant. There is

an obvious color change in the reactions carried out in the microcetrifuge tubes. While

unmodified RNase A in solution is colorless, the first micorcentrifuge tube (T1) containing no L-

ascorbic acid exhibited brown color. The brown color fades to colorless moving across the

tubes, which suggests RNase A is less modified in the microcentrifuge tubes to the right. The

48

microcentrifuge tubes to the right contained an increasing concentration of L-ascorbic acid. The

SDS-PAGE gel data shown in Figure 3.8 confirms this idea. Moving from left to right across

this gel, the concentration of L-ascorbic acid increases and the extent of modification of RNase

A decreases. Lane 2 (L2) contained no L-ascorbic acid and shows modified RNase A. Lane 4

(L4) contains equal concentrations of L-ascorbic acid and ClpBQ and shows almost all

modification of RNase A by ClpBQ inhibited. Lanes 5 and 6 (L5 and L6) contained L-ascorbic

acid in excess of ClpBQ and show no obvious modification of RNase A.

3.2.6 Detection of Lysine Oxidized by ClpBQ

Unmodified RNase A is clear in solution and has a very stable absorbance profile.

Figure 3-9 shows a UV/Vis spectrum of RNase A in a pH 7.0 phosphate buffer. There is a

characteristic λmax at 280 nm for unmodified RNase A.

Once RNase A is treated with ClpBQ, several changes are easily observed. The most

obvious change is the color change of the solution. Upon incubation with ClpBQ, RNase A is

brown in solution. Furthermore, the absorbance characteristics changed. A UV/Vis spectrum of

RNase A modified by ClpBQ for 1, 24, 48, and 72 hr, respectively, is shown in Figure 3.10.

These spectra differ significantly from Figure 3.9, which shows unmodified RNase A. This

suggests that the structure of RNase A has been altered.

2,4-dinitrophenylhydrazine (DNP) is a common reagent for trapping ketones and

aldehydes. Natural unmodified proteins contain no ketones or aldehydes present in their

structure. However, if lysine is oxidized, then it will be converted to an aldehyde. Thus DNP

would be able to bind modified RNase A by reacting with the modified lysine. DNP is detected

by UV/Vis as a peak from 350 nm – 450 nm. As shown in Figure 3.10, there is no peak from

49

350 nm – 450 nm. Figure 3.11 is a UV/Vis spectrum of DNP treated modified RNase A.

Excess DNP was removed by spin column before this spectrum was generated. In each sample,

a characteristic peak was detected at 360 nm. This suggests DNP has successfully bound to

modified RNase A which supports the idea that modified RNase A contains oxidized lysine.

50

3.3 CONCLUSIONS FROM PROTEIN MODEL REACTIONS

In conclusion, RNase A can be modified by ClpBQ at physiological pH and temperature.

This has been shown by SDS-PAGE. Furthermore, there is a direct relationship between the

degree of modification of RNase A and the concentration of ClpBQ in solution. A greater

concentration of ClpBQ in solution will result in RNase A being modified to a greater extent in a

given amount of time. Similarly, there is a direct relationship between the degree of

modification of RNase A and the incubation time with ClpBQ. Generally speaking, a longer

incubation time will result in RNase A being modified to a greater extent, however at some time

point modification will cease. This “stop” modification time point is dependent on the

concentration of ClpBQ in solution.

The modification of RNase A we observed is consistent with protein crosslinking. The

results elucidated from DAB modification of RNase A and ClpBQ modification of RNase A

were similar suggesting ClpBQ induces protein crosslinking.

Modification of RNase A can be inhibited by the reducing compounds NADH and L

ascorbic acid. However, L ascorbic acid inhibits the modification of RNase A more efficiently

than NADH at equal concentrations.

ClpBQ does oxidize lysine generating an aldehyde. This was determined by trapping the

aldehyde with DNP and detecting the unique spectral feature characteristic of a DNP derivative

using UV/Vis spectroscopy.

Figure 3.1: SDS-PAGE of variable concentrations of Cl

MM (Molecular Marker): 18.3; 28; 39.2; 60; 84; 120; 215 kDa

L 1: Control RNase A

L 2: Control RNase A (TCA

L 3: 0.1 mM ClpBQ

L 4: 0.5 mM ClpBQ

L 5: 1.0 mM ClpBQ

L 6: 5.0 mM ClpBQ

L 7: 10.0 mM ClpBQ

MM L 1

PAGE of variable concentrations of ClpBQ with RNase A

MM (Molecular Marker): 18.3; 28; 39.2; 60; 84; 120; 215 kDa (Data Duplicated)

L 2: Control RNase A (TCA Precipitation)

L 2 L 3 L 4 L 5 L 6 L 7

51

(Data Duplicated)

52

Figure 3.2: SDS-PAGE of 1.0 mM DAB with RNase A

MM (Molecular Marker): 18.3; 28; 39.2; 60; 84; 120; 215 kDa (Data Duplicated)


*L 2-7 all contain RNase A and 1.0 mM DAB

L 2: Wash-Acetone; Resuspension-Electrophoresis Buffer

L 3: Wash-Acetone; Resuspension-Phosphate Buffer

L 4: Wash-Electrophoresis Buffer; Resuspension-Electrophoresis Buffer

L 5: Wash-Electrophoresis Buffer; Resuspension-Phosphate Buffer

L 6: Wash-Phosphate Buffer; Resuspension-Electrophoresis Buffer

L 7: Wash-Phosphate Buffer; Resuspension-Phosphate Buffer

MM L 1 L 2 L 3 L4 L 5 L 6 L 7

Figure 3.3(A/B): SDS-PAGE of 5.0 mM



L 2: 10 minute incubation; 37°C

L 3: 20 minute incubation






L 2: 1 hour incubation; 37°C





MM L 1

MM L

(A)

(B)

PAGE of 5.0 mM ClpBQ with RNase A (Data Duplicated)













1 L 2 L 3 L 4 L 5 L 6

L 1 L 2 L 3 L 4 L 5 L 6

53

BQ with RNase A (Data Duplicated)

54

Figure 3.4(A/B): SDS-PAGE of 1.0 mM ClpBQ with RNase A (Data Duplicated)















MM L 1 L 2 L 3 L 4 L 5 L 6

MM L 1 L 2 L 3 L 4 L 5 L 6

(A)

(B)

Figure 3.5(A/B): SDS-PAGE of 0.5 mM Cl

MM (Molecular Marker):














MM L

MM L 1 L

(A)

(B)

PAGE of 0.5 mM ClpBQ with RNase A (Data Duplicated)













MM L 1 L 2 L 3 L 4 L 5 L 6

L 1 L 2 L 3 L 4 L 5 L 6

55

BQ with RNase A (Data Duplicated)

Figure 3.6: SDS-PAGE of ClpBQ modification of RNase A inhibited by NADH (Data

Duplicated)



L 2: 5.0 mM ClpBQ; 0.0 mM NADH





MM L

BQ modification of RNase A inhibited by NADH (Data


BQ; 0.0 mM NADH

BQ; 1.0 mM NADH

BQ; 5.0 mM NADH

BQ; 10.0 mM NADH

BQ; 50.0 mM NADH

L 1 L 2 L 3 L 4 L 5 L 6

56

BQ modification of RNase A inhibited by NADH (Data

Figure 3.7: Photo of microcentrifuge tubes; tubes contain a series of incubation reactions of

ClpBQ, RNase A, and L ascorbic acid. Reactions are in phosphate buffer (50 mM, pH 7.0) at 37

°C (Data Duplicated)

T 1: 5.0 mM ClpBQ; 0.0 mM L Ascorbic Acid





Figure 3.8: SDS-PAGE of ClpBQ modification of RNase A inhibited by L Ascorbic Acid



L 2: 5.0 mM ClpBQ; 0.0 mM L Ascorbic Acid


L 4: 5.0 mM ClpBQ; 5.0 mM L Ascorb



MM L 1

T 1 T

Photo of microcentrifuge tubes; tubes contain a series of incubation reactions of

BQ, RNase A, and L ascorbic acid. Reactions are in phosphate buffer (50 mM, pH 7.0) at 37

BQ; 0.0 mM L Ascorbic Acid





BQ modification of RNase A inhibited by L Ascorbic Acid







1 L 2 L 3 L 4 L 5 L 6

T 2 T 3 T 4 T 5

57

Photo of microcentrifuge tubes; tubes contain a series of incubation reactions of

BQ, RNase A, and L ascorbic acid. Reactions are in phosphate buffer (50 mM, pH 7.0) at 37

BQ modification of RNase A inhibited by L Ascorbic Acid

58

Figure 3.8: A representative UV/Vis spectrum of control RNase A (0.5 mg/ml) in phosphate

buffer (50 mM; pH 7.0)

Unmodified RNase A

59

Figure 3.9: Averaged UV/Vis spectra of modified RNase A incubated with ClpBQ at variable

time periods; 1, 24, 48, 72 hr.

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

200 300 400 500 600 700

Absorbance

Wavelength (nm)

RNase A modified by ClpBQ

1 hour

24 hours

48 hours

72 hours

60

Figure 3.10: Averaged UV/Vis spectra for ClpBQ modified RNase A treated with DNP.

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

200 300 400 500 600 700

Absorbance

Wavelength (nm)

DNP treatment of ClpBQ modified RNase A

1 hour

24 hours

48 hours

72 hours

360 nm

61

3.4 EXPERIMENTAL

3.4.0 SDS-PAGE Protocols

Lower Gel 4x Buffer; 1.5 M tris at pH 8.8:

18.2 g Tris base was added to 70 mL of Millipore water in a 100 mL beaker. The pH was

adjusted to 8.8 using HCl and NaOH. Then the solution was brought to the final volume of 100

mL in a 100 mL volumetric flask using Millipore water. Solution was stored at 4 ºC.

Upper Gel 4x Buffer; 0.5 M tris at pH 6.8:

6.1 g Tris base was added to 70 mL of Millipore water in a 100 mL beaker. The pH was

adjusted to 6.8 using HCl and NaOH. Then the solution was brought to the final volume of 100

mL in a 100 mL volumetric flask using Millipore water. Solution was stored at 4 ºC.

40% Acrylamide/Bis Solution:

9.7 g acrylamide and 0.3 g bis-acrylamide were added to a 50 mL centrifuge tube.

Solution was brought to a final volume of 25 mL using Millipore water. The mixture residue

was heated in a microwave to solubilize. Solution was stored at 4 ºC.

N,N,N’,N’-Tetramethylethylenediamine (TEMED):

TEMED was purchased as a liquid. It was stored at 4 ºC.

10% Sodium Dodecyl Sulfate (SDS):

2.0 g of SDS was added to 18.0 mL of Millipore water in a 50 mL centrifuge tube. The

residue was solubilized via vortexing. Solution was stored at room temperature.

10% Ammonium Persulfate (APS):

2.0 g of APS was added to 18.0 mL of Millipore water in a 50 mL centrifuge tube. The

residue was vortexed in order to dissolve APS. Since APS in solution is air-sensitive, 0.5 mL

62

aliquots were taken from the stock solution and divided into microcentrifuge tubes. These tubes

were flushed with Ar gas and stored at -80 ºC.

Loading Dye:

To a 25 mL centrifuge tube was added 1.0 mL Tris/HCl Buffer (0.5 M), 4.0 mL Glycerol

(50% by weight), 0.250 mL bromophenol blue (2%; 2.0 mg in 8.0 mL ethanol), 0.500 mL 2-

mercaptoethanol, and 1.0 g SDS. The mixture was brought to a final volume of 10.0 mL using

Millipore water. The residue was solubilized via vortexing. 0.500 mL aliquots were divided into

microcentrifuge tubes from the stock soluiton. These tubes were flushed with Ar gas and stored

at -80 ºC.

Electrophoresis Buffer (5x):

15.0 g of Tris base and 72.0 g Glycine was added to a 1 L volumetric flask. To this

residue was added 900 mL of Millipore water and the mixture is homogenized. To the mixture

was added 5.0 g of SDS. SDS was solubilized and using Millipore water the mixture was

brought to volume. This solution was stored at room temperature.

Electrophoresis Buffer (1x):

200 mL of electrophoresis buffer (5x) was diluted with Millipore water to 1 L. This

solution was stored at room temperature.

Staining Solution:

To a large media bottle was added 1.0 g Coomassie Blue R250, 250 mL methanol, 100

mL glacial acetic acid, and 650 mL Millipore water. This solution was homogenized by shaking

and stored at room temperature.

63

Destaining Solution:

To a large media bottle is added 250 mL methanol, 100 mL glacial acetic acid, and 650

mL Millipore water. This solution was homogenized by shaking and stored at room temperature.

SDS-PAGE Gel Casting:

Electrophoresis glass plates were cleaned with 70% ethanol and air-dried. Casting

apparatus was assembled using new plastic pouches to hold glass plates and spacers. Our

laboratory used a 10% acrylamide resolving gel and a 3.2% acrylamide in all stacking gels for

SDS-PAGE. The composition of the lower gel and the upper gel can be found on Table 3.1 and

Table 3.2 respectively. 5.0 mL of resolving gel solution was loaded between the plates using a

transfer pipette and allowed to polymerize for 2 hr. Stacking gel solution was loaded to fill the

rest of space with a transfer pipette. A 10 well comb was used to cast the wells in the stacking

gel. After 45 min the gels were fully polymerized. They were stored at 4 ºC.

SDS-PAGE Preparation:

Plates were loaded into gel holding apparatus. Electrophoresis buffer (1x) was added to

the apparatus until the anode and cathode were completely submerged and the wells of the gel

were submerged. Samples containing proteins of interest were loaded into the wells via pipette.

The voltage of the system was set to 100 V, and the current was set to 30.0 mA per gel. Thus if

multiple gels were run then the current was set equal to 30.0 mA times the number of gels run.

Electrophoresis was carried out for approximately 2.5 hr.

Staining and Destaining of Gel:

Gels are removed from the electrophoresis apparatus and the stacking gel was discarded.

The resolving gel was placed in a staining solution and set on a shaker table. After

approximately 2 hr of staining, the gel was removed and washed with Millipore water. The gel

64

was placed in a destaining solution containing Kimwipes and put back on the shaker table.

Every 30 min, the Kimwipes were removed and fresh Kimwipes were added to the solution.

Destaining will last between 2 and 4 hr. After destaining, the gels were washed with Millipore

water and scanned as JPEG files.

Protein Purification via TCA Precipitation:

To purify a sample containing protein, this lab used trichloroacetic acid (TCA)

precipitation. This method causes protein to precipitate out of solution. Cold TCA was added to

a sample and the sample was incubated on ice for 20 min. Following incubation, the sample was

centrifuged at 4 ºC for 20 min at 12,000 rpm. Then, the protein pellet was formed at the bottom

of the microcentrifuge tube. The supernatant was removed via pipett without disturbing the

pellet, and the pellet was washed twice with cold electrophoresis buffer (1x) unless otherwise

noted. The pellet was resuspended in electrophoresis buffer (1x) unless otherwise noted.

Sample Preparation for SDS-PAGE:

Resuspended samples containing proteins in various conditions were placed in a clean

labeled microcentrifuge tube. An equivalent volume of loading dye was added to the sample.

The solution was vortexed then heated in a boiling water bath for 5 min.

3.4.1 ClpBQ Concentration Dependent Modification of RNase A

Working in new microcentrifuge tubes, RNase A stock solution (2 mg/mL), ClpBQ stock

solution (either 100 mM or 10 mM), and phosphate buffer (pH 7.0; 50 mM) were combined

according to Table 3.3. All samples were incubated at 37 ºC for 1 hr. Samples containing

proteins were then purified by TCA precipitation. SDS-PAGE data were obtained for all protein

samples.

65

Note on Reagents:

100 mM ClpBQ was prepared by dissolving 14.3 mg of ClpBQ in 1.0 mL of MeOH and

10 mM ClpBQ was made by a 10-fold dilution of 100 mM ClpBQ using MeOH.

3.4.2 1,2-Diacetylbenzene Dependent Modification of RNase A

RNase A was modified with 1,2-diacetylbenzene (1.0 mM). Working in new

microcentrifuge tubes, RNase A stock solution (20 mg/mL), 1,2-diacetylbenzene (50 mM) stock

solution, and phosphate puffer (pH 7.0; 50 mM) were combined according to Table 3.4. All

samples were incubated at 37 ºC for 1 hr. Samples containing proteins were purified by TCA

precipitation. For the wash, the sample pellets were gently washed twice with 50 µL of the

solution indicated in Table 3.4. 25 µL of buffer indicated in Table 3.4 was used to resuspend

the pellets by vortexing. SDS-PAGE data were obtained for all protein samples.

Note on Reagents:

50 mM 1,2-diacetylbenzene was prepared by solubilizing 8.1 mg of 1,2-diacetylbenzene

in 1.0 mL MeOH.

3.4.3 ClpBQ Time and Concentration Dependent Modification of RNase A

RNase A was modified with ClpBQ (5.0, 1.0 and 0.5 mM) and incubated for variable

times. The times evaluated in this experiment are 10, 20, 30, 40, and 50 min, 1, 2, 3, 4 and 5 hr.

RNase A stock solution (20 mg/mL), ClpBQ stock solution, and phosphate buffer (pH

7.0; 50 mM) were combined in new microcentrifuge tubes on ice according to Tables 3.5(A/B),

3.6(A/B), and 3.7(A/B). All samples were incubated at 37 ºC for the time indicated in Tables

66

3.5(A/B), 3.6(A/B), and 3.7(A/B). Samples containing protein were purified by TCA

precipitation. SDS-PAGE data were obtained for all protein samples.

3.4.4 ClpBQ Modification of RNase A Inhibited by Concentration Dependent NADH

Fresh NADH (100 mM) and ClpBQ (100 mM) solutions were prepared and put on ice.

RNase A stock solution (2.0 mg/mL) phosphate buffer (pH 7.0; 50 mM), NADH stock solution

(100 mM) and ClpBQ stock solution (100 mM) were combined in new microcentrifuge tubes on

ice according to Table 3.8 and vortexed. All samples were incubated at 37 ºC for 1 hour.

Samples containing protein were purified by TCA precipitation. SDS-PAGE data were obtained

for all protein samples.

3.4.5 ClpBQ Modification of RNase A Inhibited by Concentration Dependent L-Ascorbic

Acid

Fresh L-ascorbic acid (100 mM) and ClpBQ (100 mM) solutions were prepared and put

on ice. RNase A stock solution (2.0 mg/mL) phosphate buffer (pH 7.0; 50 mM), L-ascorbic acid

stock solution (100 mM) and ClpBQ stock solution (100 mM) were combined in new

microcentrifuge tubes on ice according to Table 3.9 and vortexed. All samples were incubated

at 37 ºC for 1 hour. Samples containing protein were purified by TCA precipitation. SDS-

PAGE data were obtained for all protein samples.

3.4.6 DNP Treatment of ClpBQ Modified RNase A

Modified RNase A samples were obtained from fellow researcher Caitlin Redman—four

samples were obtained in triplicate; each sample contained purified modified RNase A. The

67

RNase A was modified with 1.0 mM ClpBQ. Incubation periods were 1, 24, 48, and 72 hr.

Following incubation, samples were purified by dialysis then frozen at -80 ºC until this

experiment was run.

To new microcentrifuge tubes was added, 0.25 mL of each sample, and 0.25 mL DNP-

HCl (10 mM). These samples were incubated at room temperature for 30 min. Samples were

loaded into Millipore spin columns (pore size < 3,000 Da) and centrifuged at 12,000 rpm for 2

hr. Following this initial spin, 200 µL of Millipore water was added to the top of each spin

column and samples were centrifuged at 12,000 rpm for 1 hr to wash off excess DNP. To

recover the protein, spin columns were inverted over new microcentrifuge tubes and centrifuged

at 1000 rpm for 3 min.

The spinout was added to 3.0 mL of Guanidine (6.0 M) for UV/Vis spectroscopy.

68

Table 3.1: Composition of Resolving Gel (10% acrylamdie/bis) for SDS-PAGE

Lower Gel 4x buffer 5 mL

Millipore Water 7.8 mL

Acrylamide/Bis (40%) 5 mL

SDS (10%) 2 mL

APS (10%) 100 µL

TEMED 10 µL

Table 3.2: Composition of Stacking Gel for SDS-PAGE

Upper Gel 4x buffer 2.5 mL

Millipore Water 6.6 mL

Acrylamide/Bis (40%) 0.8 mL

SDS (10%) 1.0 mL

APS (10%) 100 µL

TEMED 10 µL

69

Table 3.3 ClpBQ Concentration Dependent Modification of RNase Sample Compositions

SAMPLE NAME Control Control TCA 0.1 mM 0.5 mM 1.0 mM 5.0 mM 10.0 mM

Sample Prep Protein Stock* (µL) 25 25 25 25 25 25 25

Protein (µg) 50 50 50 50 50 50 50

100 mM ClpBQ Stock (µL) - - - - 0.5 2.5 5.0

10 mM ClpBQ Stock (µL) - - 0.5 2.0 - - -

Phosphate Buffer (µL) - 20 24.5 22.5 24.5 22.5 20

Incubation time - 1 h 1 h 1 h 1 h 1 h 1 h

Incubation temperature - 37 ºC 37 ºC 37 ºC 37 ºC 37 ºC 37 ºC

Final Volume (µL) - 50 50 50 50 50 50

TCA (20%) (µL) - 12.5 12.5 12.5 12.5 12.5 12.5

Wash (2x) Cold 1x Electrophoresis

Buffer (µL) - 50 50 50 50 50 50

Resuspension 1x Electrophoresis Buffer (µL) - 25 25 25 25 25 25

SDS-PAGE Loading dye (µL) 25 25 25 25 25 25 25

Loading volume (µL) 10 10 10 10 10 10 10

* Protein Stock concentration: 2.0 mg/mL

70

Table 3.4 1,2-Diacetylbenzene Modification of RNase A Sample Compositions

SAMPLE NAME Control E-Acetone P-Acetone E-E. Buf P-E. Buf E-P. Buf P-P. Buf

Sample Prep Protein Stock* (µL) 4 5 5 5 5 5 5

Protein (µg) 40 100 100 100 100 100 100

50 mM DAB Stock (µL) - 1 1 1 1 1 1

phosph buf (µL) - 44 44 44 44 44 44

Incub Time - 1 h 1 h 1 h 1 h 1 h 1 h

Incub Temp - 37 ºC 37 ºC 37 ºC 37 ºC 37 ºC 37ºC

Final Volume (µL) - 50 50 50 50 50 50

TCA Purificaiton

TCA (20%) (µL) 12.5 12.5 12.5 12.5 12.5 12.5

Incub Time 20 min 20 min 20 min 20 min 20 min 20 min

Incub Temp On Ice On Ice On Ice On Ice On Ice On Ice

Centrifuge Time 20 min 20 min 20 min 20 min 20 min 20 min

Centrifuge Temp 4 ºC 4 ºC 4 ºC 4 ºC 4 ºC 4 ºC

Wash (2x)

Cold Acetone (µL) 50 50 - - - -

Cold E. Buffer (µL) - - 50 50 - -

Cold P. Buffer (µL) - - - - 50 50

Resuspendion E. Buffer (µL) 16 25 - 25 - 25 -

P. Buffer (µL) - 25 - 25 - 25

*Protein stock concentration 20 mg/mL

71

Table 3.5A ClpBQ Time and Concentration Dependent Modification of RNase A; Sample Compositions for 5.0 mM ClpBQ (short)

SAMPLE NAME Control 10 min 20 min 30 min 40 min 50 min

Sample Prep Protein Stock* (µL) 2 5 5 5 5 5

Protein (µg) 40 100 100 100 100 100

100mM ClpBQ Stock (µL) - 2.5 2.5 2.5 2.5 2.5

Phosphate Buffer (µL) - 42.5 42.5 42.5 42.5 42.5

Incubation time - 10 min 20 min 30 min 40 min 50 min

Incubation temperature - 37 ºC 37 ºC 37 ºC 37 ºC 37 ºC

Final Volume (µL) 50 50 50 50 50

TCA (20%) (µL) - 12.5 12.5 12.5 12.5 12.5

Wash (2x) Cold 1x Electrophoresis Buffer (µL) - 50 50 50 50 50

Resuspension 1x Electrophoresis Buffer (µL) 18 25 25 25 25 25

SDS-PAGE Loading dye (µL) 20 25 25 25 25 25

Loading volume (µL) 10 10 10 10 10 10

Table 3.5B ClpBQ Time and Concentration Dependent Modification of RNase A; Sample Compositions for 5.0mM ClpBQ (long)

SAMPLE NAME Control 1 hr 2 hr 3 hr 4 hr 5 hr


Protein (µg) 40 100 100 100 100 100

100mM ClpBQ Stock (µL) - 2.5 2.5 2.5 2.5 2.5


Incubation time - 1 hour 2 hours 3 hours 4 hours 5 hours



TCA (20%) (µL) - 12.5 12.5 12.5 12.5 12.5






72




Protein (µg) 40 100 100 100 100 100

100mM ClpBQ Stock (µL) - 0.5 0.5 0.5 0.5 0.5





TCA (20%) (µL) - 12.5 12.5 12.5 12.5 12.5





Table 3.6B ClpBQ Time and Concentration Dependent Modification of RNase A; Sample Compositions for 1.0 mM ClpBQ (long)



Protein (µg) 40 100 100 100 100 100

100mM ClpBQ Stock (µL) - 0.5 0.5 0.5 0.5 0.5





TCA (20%) (µL) - 12.5 12.5 12.5 12.5 12.5






73




Protein (µg) 40 100 100 100 100 100

50mM ClpBQ Stock (µL) - 0.5 0.5 0.5 0.5 0.5





TCA (20%) (µL) - 12.5 12.5 12.5 12.5 12.5





Table 3.7B ClpBQ Time and Concentration Dependent Modification of RNase A; Sample Compositions for 0.5 mM ClpBQ (long)



Protein (µg) 40 100 100 100 100 100

50mM ClpBQ Stock (µL) - 0.5 0.5 0.5 0.5 0.5





TCA (20%) (µL) - 12.5 12.5 12.5 12.5 12.5






74

Table 3.8 ClpBQ Modification of RNase A Inhibited by Concentration Dependent NADH, Sample Compositions

[ClpBQ] /mM 0.0 5.0 5.0 5.0 5.0 5.0

[NADH] /mM 0.0 0.0 1.0 5.0 10.0 50.0

SAMPLE NAME Control 0.0 1.0 5.0 10.0 50.0


Protein (µg) 40 100 100 100 100 100

100mM NADH Stock (µL) - - 0.5 2.5 5.0 25

100mM ClpBQ Stock (µL) - 2.5 2.5 2.5 2.5 2.5


Incubation time - 1 hour 1 hour 1 hour 1 hour 1 hour



TCA (20%) (µL) 12.5 12.5 12.5 12.5 12.5

Wash (2x) 1x Electrophoresis Buffer (µL) 50 50 50 50 50





75

Table 3.9 ClpBQ Modification of RNase A Inhibited by Concentration Dependent L-Ascorbic Acid, Sample Compositions

[ClpBQ] /mM 0.0 5.0 5.0 5.0 5.0 5.0

[L-Ascorbic Acid] /mM 0.0 0.0 1.0 5.0 10.0 50.0

SAMPLE NAME Control 0.0 1.0 5.0 10.0 50.0


Protein (µg) 40 100 100 100 100 100

100mM L-Ascorbic Acid Stock (µL) - - 0.5 2.5 5.0 25

100mM ClpBQ Stock (µL) - 2.5 2.5 2.5 2.5 2.5


Incubation time - 1 hour 1 hour 1 hour 1 hour 1 hour



TCA (20%) (µL) 12.5 12.5 12.5 12.5 12.5

Wash (2x) 1x Electrophoresis Buffer (µL) 50 50 50 50 50





76

APPENDIX A

EFFECTS OF METHANOL ON RNASE A MODIFICATION

77

A.1 BACKGROUND

At high concentrations (about 100 mM) ClpBQ was not soluble in water, thus to put

ClpBQ in solution, methanol (MeOH) was used. Since MeOH, like ClpBQ, is foreign to

biological systems, there is a possibility it is MeOH which is modifying protein and no ClpBQ.

To test this idea, we examined the effects of MeOH on RNase A. The overall goal of this

experiment was to see if MeOH causes any modification to RNase A.

5.0% MeOH by volume was the highest concentration of MeOH ever present in our

experiments. We tested the effects 1.0, 2.5, 5.0, 7.5, and 10.0% MeOH by volume has on RNase

A.

78

A.2 RESULTS AND DISCUSSION

The effects of MeOH on RNase A modification were examined and the SDS-PAGE data

are shown in Figure A. Moving from left to right across the gel, the concentration of MeOH in

solution increases from 0% to 10.0% while all other variables are constant. Lane 1 (L1) shows

control RNase A. There are two visible bands in this lane. The first represents unmodified

RNase A, this is the more intense band farther down the gel, and the second most likely reflects

an impurity in the stock RNase A solution, since we were not working with 100% pure RNase A.

This is the band above the unmodified RNase A and is much less intense. Lanes 2, 3, 4, 5, and 6

(L2, L3, L4, L5, and L6) all show the same bands which are present in L1 with no additional

bands present. Also the intensity of all bands is the same in all the lanes.

It should also be noted, upon incubation with MeOH, solutions containing RNase A

remained colorless. Recall unmodified RNase A in solution is also colorless.

Put simply, the SDS-PAGE data show no difference among any of the lanes. The control

RNase A looks the exact same as the RNase A incubated with 10% MeOH. This information

coupled with the fact that the reaction tubes did not become brown following incubation suggests

MeOH does not modify RNase A under the reaction conditions we have examined, and any

modification to RNase A was most likely caused by other chemical species in solution.

79

Figure A: SDS-PAGE of MeOH modification of RNase A



L 2: 1.0% MeOH

L 3: 2.5% MeOH

L 4: 5.0% MeOH (highest concentration of MeOH in all experiments)

L 5: 7.5% MeOH

L 6: 10.0% MeOH

MM L 1 L 2 L 3 L4 L 5 L 6

80

A.3 EXPERIMENTAL

RNase A stock solution (20 mg/mL), phosphate buffer (pH 7.0; 50 mM) and MeOH were

combined in new microcentrifuge tubes on ice according to Table A and vortexed. All samples

were incubated at 37 ºC for 1 hour. Samples containing protein were purified by TCA

precipitation. SDS-PAGE data were obtained for all protein samples.

81

Table A Composition of samples for effects of methanol on RNase A modificiaiton. Concentration of methanol in solution is based

on percent volume.

SAMPLE NAME Control 1.0% 2.5% 5.0% 7.5% 10.0%


Protein (µg) 100 100 100 100 100 100

Methanol (µL) - 0.5 1.3 2.5 3.8 5.0


Incubation time - 1 h 1 h 1 h 1 h 1 h


Final Volume (µL) - 50 50 50 50 50

TCA (20%) (µL) - 12.5 12.5 12.5 12.5 12.5

Wash (2x) Cold 1x Electrophoresis

Buffer (µL) - 50 50 50 50 50




* Protein Concentration (20 mg/mL)

82

ACKNOWLEDGEMENTS

Department:

UTC Chemistry Department

Thanks for all the encouragement, patience and perpetual advice. I will never forget my

time here.

Individuals:

Advisor: Dr. Jisook Kim

This would have never happened without you! Thanks so much

for taking a chance and believing in me. Good luck with the new

addition to your family!

Fellow researcher: Min Jung Kang

Fellow researcher: Seven Ledford

Fellow researcher: Caitlin Redman

Special thanks for providing RNase A spectrum, Figure 3.8

Collaborator: Dr. Titus Albu (Tennessee Tech)

DHON Examiner: Dr. Tom Rybolt

DHON Examiner: Dr. John Lynch

DHON Liaison: Dr. Kathleen Wheatley

Funding:

Grote Chemistry Fund

Provost Student Research Award

UC Foundation Fund: Start-up and Strategic Initiative

83

BIBLIOGRAPHY

Atkins, P.; Paula, J. Atkins' Physical Chemistry; Eighth Edition. New York, NY : W. H. Freeman

and Company, 2006.

Benner, Bruce A Jr.; Brynre, Nelson P.; Wise, Stephen A.; Mulholland, George W.; Lao, Robert

C.; Fingas, Mervin F. Polycyclic aromatic hydrocarbon emissions from the combustion of

crude oil on water. Environ. Sci. Technol. 1990, 24, 1418-1427.

Bolton, J.; Trush, M.; Penning, T.; Dyryhurst, G.; Monks, T. Role of Quinones in Toxicology,

Chem. Res. Toxicol. 2000, 13, 135-160.

Boyer, R. Concepts in Biochemistry; Third Edition. Hoboken, NJ. John Wiley & Sons. Inc.,

2006.

Brady, J. E.; Senese, F. Chemistry Matter and Its Changes; Fourth Edition. Hoboken, NJ : John

Wiley & Sons Inc., 2004.

Bruice, P. Y., Organic Chemistry; Fifth Edition. Upper Saddle River, NJ. Pearson Prentice Hall,

2007.

Chenna, A.; Hang, B.; Rydberg, B.; Kim, E.; Pongracz, K.; Bodell, W. J.; Singer, B. The benzene

metabolite p-benzoquinone forms adducts with DNA bases that are excised by a repair

activity from human cells that differs from an ethenoadenine glycosylase, Proc Natl Acad

Sci USA 1995, 92, 5890-5894.

Ding, Y.; Ashley, D.; Watson, C. Determination of 10 Carcinogenic Polycyclic Aromatic

Hydrocarbons in Mainstream Cigarette Smoke. J. Agric, Food Chem. 2007, 55, 5966-

5973.

Dong, S.; Fu, P.; Shirsat, R.; Hwang, H.; Leszczynski, J.; Yu, H. UVA Light-Inducted DNA

Cleavage by Isomeric Methylbenz[a]anthracenes, Chem. Res. Toxicol. 2002, 15, 400-

407.

Eilstein J., Giménez-Arnau, E., Duché, D., Rousset, Françoise. Mechanistic Studies on the

Lysine-Induced N-Formylation of 2,5-Dimethyl-p-benzoquinonediimine, Chem. Res.

Toxicol 2007, 20, 1155-1161.

Fisher, A. A.; Labenski, M. T.; Malladi, S.; Gokhale, V.; Bowen, M. E.; Milleron, R. S.; Bratton,

S. B.; Monks, T. J.; Lau, S. S. Quinone electrophiles selectively adduct “electrophile

binding motifs” within cytochrome c, Biochemistry 2007, 46, 11090-11100.

Garrett, R.; Guenette, C.; Haith, C.; Prince, R.; Pyrogenic Polycyclic Aromatic Hydrocarbons in

Oil Burn Residues, Environ. Sci. Technol. 2000, 34, 1934-1937.

84

Giger, W.; Blurner, M. Polycyclic aromatic hydrocarbons in the environment. Isolation and

characterization by chromatography, visible, ultraviolet, and mass spectrometry, Anal.

Chem. 1974, 46, 1663-1671.

Gribbin, John. The Scientists. (2002) New York, NY. Random House

Hanzlik, R. P.; Harriman, S. P.; Frauenhoff, M. M. Covalent binding of benzoquinone to reduced

ribonuclease. Adduct structures and stoichiometry, Chem Res Toxicol 1994, 7, 177-184.

Kim, M. S., Hashemi, S. B., Spencer, P. S., and Sabri, M. I. (2002) Amino acid and protein

targets of 1,2-diacetylbenzene, a potent aromatic gamma-diketone that induces proximal

neurofilamentous axonopathy, Toxicol Appl Pharmacol 183, 55-65

Kondrová, E.; Stopka, P.; and Souček, P. Cytochrome P450 destruction benzene metabolites 1,4-

benoquinone and 1,4-hydroquinone and the formation of hydroxyl radicals in minipig

liver microsomes, Toxicol in Vitro 2007, 21, 566-575.

Levine, R. L.; Williams, J. A.; Stadtman, E. R.; Shacter, E. Carbonyl assays for determination of

oxidatively modified proteins, Methods Enzymol 1994, 233, 346-357

Mason, D.; Liebler, D. Characterization of Benzoquinone—Peptide Adducts by Electrospray

Mass Spectrometry, Chem. Res. Toxicol. 2000, 13, 976-982.

McCracken, P.; Bolton, J.; Thatcher, G. Covalent Modificaiton of Proteins and Peptides by the

Qunone Methide from 2-tert Butyl-4,6-dimethylphenol: Selectivity and Reactivity with

Respect to Competitive Hydration, J. Org. Chem. 1997, 62, 1820-1825.

McLean, M. R.; Bauer, U.; Amaro, A. R.; Robertson, L. W. Identification of catechol and

hydroquinone metabolites of 4-monochlorobiphenyl, Chem Res Toxicol 1996, 9, 158-

164.

Nakayama, A.; Kawanishi, M.; Takebe, H.; Morisawa, S.; Yagi, T. Molecular analysis of

mutations induced by a benzene metabolite, p-benzoquinone, in mouse cells using a novel

shuttle vector plasmid, Mutat Res 1999, 444, 123-131.

Osborne, R.; Raner, G.; Hager, L.; Dawson, J. C. funago Chloroperoxidase is also a

Dehaloperoxidase: Oxidative Dehalogenation of Halophenols, J. Am. Chem. Soc. 2006,

128, 1036-1037.

Porter Roy. The Greatest Benefit to Mankind; A Medical History of Humanity. (1997) New

York, NY. W. W. Norton & Company.

Skoog, D. A.; Holler, F. J.; Crouch, S. R. Principles of Instrumental Analysis; Seventh Edition.

Belmont, CA : Thomson Higher Education, 2007.

85

Snyder, R.; Hedli, C. C. An Overview of Benzene Metabolism, Environ Health Perspet 1996, 104

(Suppl. 6), 1165-1172.

Waidyanatha, S.; Sangaiah, R.; Rappaport, S. M. Characterization and Quantification of

Cysteinyl Adducts of Benzene Diol Epoxide. Chem. Res. Toxicol, 2005, 18, 1178-1185.

Wang, X.; Thomas, B.; Sachdeva, R.; Arterburn, L.; Frye, L.; Hatcher, P.; Cornwell, D.; Ma, J.

Mechanism of arylating quinone toxicity involving Michael adduct formation and

induction of endoplasmic reticulum stress, PNAS. 2006, 103, 3604-3609.

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