Purification of a New Human Carbonyl Reductase - Report

8/14/2019 Purification of a New Human Carbonyl Reductase - Report

1/34

Charles University in PragueFaculty of Pharmacy in Hradec KrlovDepartment of Biochemical Sciences

Purification of a new human

carbonyl reductase

(report from the Erasmus study stay)

Jos Eduardo Oliveira da Silva Almeida

Hradec Krlov, 2008


2/34

Index

1. Theoretical part..4

1.1. Aim of the study.4

1.2. Protein Purification....4

1.2.1. Strategies5

1.2.1.1. Three phases strategy..5

1.2.2. Evaluating purification yield.....6

1.2.3. Sample preparation...7

1.2.3.1. Extraction.7

1.2.3.2. Precipitation and differential solubilization.....7

1.2.3.3. Ultracentrifugation..8

1.2.4. Chromatographic methods..8

1.2.4.1. Size exclusion chromatography..8

1.2.4.2. Ion exchange chromatography...9

1.2.4.3. Affinity chromatography.10

1.2.4.4. Hydrophobic interaction chromatography...10

1.2.4.5. HPLC....12

1.2.5. Concentration of the purified protein...12

1.2.6. Electrophoresis...12

1.3. Metabolism of carbonyl groups.....13

1.3.1. Carbonyl reducting enzymes....14

1.3.1.1. Aldoketo reductase superfamily (AKR)16

1.3.1.2. Short-chain dehydrogenases/reductases (SDR)..16

1.4. Oracin...18

2
http://en.wikipedia.org/w/index.php?title=Protein_purification&printable=yes#Strategies%23Strategieshttp://en.wikipedia.org/w/index.php?title=Protein_purification&printable=yes#Evaluating_purification_yield%23Evaluating_purification_yieldhttp://en.wikipedia.org/w/index.php?title=Protein_purification&printable=yes#Extraction%23Extractionhttp://en.wikipedia.org/w/index.php?title=Protein_purification&printable=yes#Precipitation_and_differential_solubilization%23Precipitation_and_differential_solubilizationhttp://en.wikipedia.org/w/index.php?title=Protein_purification&printable=yes#Ultracentrifugation%23Ultracentrifugationhttp://en.wikipedia.org/w/index.php?title=Protein_purification&printable=yes#Chromatographic_methods%23Chromatographic_methodshttp://en.wikipedia.org/w/index.php?title=Protein_purification&printable=yes#Size_exclusion_chromatography%23Size_exclusion_chromatographyhttp://en.wikipedia.org/w/index.php?title=Protein_purification&printable=yes#Ion_exchange_chromatography%23Ion_exchange_chromatographyhttp://en.wikipedia.org/w/index.php?title=Protein_purification&printable=yes#Affinity_chromatography%23Affinity_chromatographyhttp://en.wikipedia.org/w/index.php?title=Protein_purification&printable=yes#HPLC%23HPLChttp://en.wikipedia.org/w/index.php?title=Protein_purification&printable=yes#Concentration_of_the_purified_protein%23Concentration_of_the_purified_proteinhttp://en.wikipedia.org/w/index.php?title=Protein_purification&printable=yes#Strategies%23Strategieshttp://en.wikipedia.org/w/index.php?title=Protein_purification&printable=yes#Evaluating_purification_yield%23Evaluating_purification_yieldhttp://en.wikipedia.org/w/index.php?title=Protein_purification&printable=yes#Extraction%23Extractionhttp://en.wikipedia.org/w/index.php?title=Protein_purification&printable=yes#Precipitation_and_differential_solubilization%23Precipitation_and_differential_solubilizationhttp://en.wikipedia.org/w/index.php?title=Protein_purification&printable=yes#Ultracentrifugation%23Ultracentrifugationhttp://en.wikipedia.org/w/index.php?title=Protein_purification&printable=yes#Chromatographic_methods%23Chromatographic_methodshttp://en.wikipedia.org/w/index.php?title=Protein_purification&printable=yes#Size_exclusion_chromatography%23Size_exclusion_chromatographyhttp://en.wikipedia.org/w/index.php?title=Protein_purification&printable=yes#Ion_exchange_chromatography%23Ion_exchange_chromatographyhttp://en.wikipedia.org/w/index.php?title=Protein_purification&printable=yes#Affinity_chromatography%23Affinity_chromatographyhttp://en.wikipedia.org/w/index.php?title=Protein_purification&printable=yes#HPLC%23HPLChttp://en.wikipedia.org/w/index.php?title=Protein_purification&printable=yes#Concentration_of_the_purified_protein%23Concentration_of_the_purified_protein


3/34


4/34

1. Theoretical part

1.1. Aim of research project

This study is a part of a research project about purification of a new carbonylreductase from human liver microsomes, which participate in biotransformationof xenobiotics. Although it is a complex project, working group of Prof. V. Wslon Department of Biochemical Sciences has been involved in study ofmetabolism of xenobiotics for a long time and has considerable experience inthis area.

Originally or group was engaged with study of a anticancer drug Oracinand its biotransformation. Besides cytosolic enzymes from AKR1C family alsomicrosomal 11-hydroxysteroid dehydrogenase 1 (11-HSD1) participates in itsmetabolism. Human purified 11-HSD1 reduces Oracin in stereospecificmanner, preferably is formed (-)-DHO enantiomer (76 %) . It has not beenreported other microsomal enzymes in its metabolism. This study wasmotivated by the fact that whole human liver microsomes reduce Oracin to DHOin the rate of (-)-DHO and (+)-DHO 60:40 [13].This difference in metabolism ledus to idea that in microsomes must be at least one another enzyme participatingin metabolism of Oracin. This enzyme must reduce oracin preferably to (+)-

DHO. The aim of this study is to purify and isolate this unknown carbonylreductase from human liver microsomes. I participate in this project and I try tolearn methods that are applied in purification of enzymes.

1.2. Protein Purification

The development of techniques and methods for protein purification has

been an essential pre-requisite for many of the advancements made inbiotechnology [1].Protein purification is a series of processes intended to isolate a single

type of protein from a complex mixture. Protein purification is vital for thecharacterisation of the function, structure and interactions of the protein ofinterest. The starting material is usually a biological tissue or a microbial culture.The various steps in the purification process may free the protein from a matrixthat confines it, separate the protein and non-protein parts of the mixture, andfinally separate the desired protein from all other proteins. Separation of oneprotein from all others is typically the most laborious aspect of proteinpurification. Separation steps exploit differences in protein size, physico-

chemical properties and binding affinity [2].

4
http://en.wikipedia.org/wiki/Proteinhttp://en.wikipedia.org/wiki/Biological_tissuehttp://en.wikipedia.org/wiki/Proteinhttp://en.wikipedia.org/wiki/Biological_tissue


5/34

1.2.1. Strategies

Development of purification strategy is very complicated. It is necessary

use a systematic approach. First step is answering several question as: What isthe intended use of product? What kind of starting material is available? Whathas to be removed from purified protein? What are the economical constraints?And other similar question. It is required to obtain a protein efficiently,economically, and in sufficient purity and quantity [1].

The choice of a starting material is a key to the design of a purificationprocess. Starting material may be microbial culture, plant or animal tissues,human tissues etc. In a plant or animal, a particular protein usually is notdistributed homogeneously throughout the body; different organs or tissueshave higher or lower concentrations of the protein. Use of only the tissues ororgans with the highest concentration decreases the volumes needed toproduce a given amount of purified protein. If the protein is present in lowabundance, or if it has a high value, scientists may use recombinant DNAtechnology to develop cells that will produce large quantities of the desiredprotein (this is known as an expression system). Recombinant expressionallows the protein to be tagged, e.g. by a His-tag, to facilitate purification, whichmeans that the purification can be done in fewer steps. In addition, recombinantexpression usually starts with a higher fraction of the desired protein than ispresent in a natural source [2].

1.2.1.1. Three Phase Strategy

This strategy is used as an aid to the development of purification processfor a protein. In the Three Phase Strategy specific objectives are assigned toeach step within the process: i) in the capture phase the objectives are toisolate, concentrate and stabilise the target product, ii) during the intermediate

purificationphase the objective is to remove most of the bulk impurities such asother proteins and nucleic acids, endotoxins and viruses, and iii) in the polishing

phase the objective is to achieve high purity by removing any remaining traceimpurities or closely related substances.

5
http://en.wikipedia.org/wiki/Recombinant_DNAhttp://en.wikipedia.org/wiki/Expression_systemhttp://en.wikipedia.org/wiki/His-taghttp://en.wikipedia.org/wiki/Recombinant_DNAhttp://en.wikipedia.org/wiki/Expression_systemhttp://en.wikipedia.org/wiki/His-tag


6/34

Figure 1: Preparation and Three Phase Purification strategy [1].The selection and optimum combination of purification techniques for

Capture, Intermediate Purification and Polishing is crucial to ensure fast methoddevelopment, a shorter time to pure product and good economy. It should benoted that this strategy does not mean that all purification strategies must havethree purification steps. For example capture and intermediate purification canbe achieved in single step.

The final purification process should ideally consist of samplepreparation, including extraction and clarification when required, followed byseveral major purification steps. The number of steps used will always dependupon the purity requirements and intended use for the protein. But it isnecessary keep in mind that each purification step will cause loss of proteinproduct.For example, if a yield of 80% in each step is assumed, this will bereduced to only 20% overall yield after 8 processing steps. Consequently, itsimportant to reach the targets for yield and purity with the minimum number ofsteps and the simplestpossible design. Techniques should be organised in alogical sequence to avoid the need for conditioning steps. Product from onetechnique should be in condition suitable to the next step without any handlingsample [1].

1.2.2. Evaluating purification yield

It is necessary to develop fast and reliable analytical assay is essential tofollow the progress of purification of sample and asses of effectiveness (yield,biological activity, recovery). If the protein has a distinguishing spectroscopicfeature or an enzymatic activity, this property can be used to detect and quantifythe specific protein, and thus to select the fractions of the separation, thatcontains the protein. If antibodies against the protein are available then westernblotting and ELISA can specifically detect and quantify the amount of desiredprotein. Some proteins function as receptors and can be detected duringpurification steps by a ligand binding assay, often using a radioactive ligand [1].

The most general method to monitor the purification process is byrunning a SDS-PAGE of the different steps. This method only gives a rough

6
http://en.wikipedia.org/wiki/Spectroscopyhttp://en.wikipedia.org/wiki/Enzymehttp://en.wikipedia.org/wiki/Western_blottinghttp://en.wikipedia.org/wiki/Western_blottinghttp://en.wikipedia.org/wiki/ELISAhttp://en.wikipedia.org/wiki/Receptor_(biochemistry)http://en.wikipedia.org/wiki/Ligand_(biochemistry)http://en.wikipedia.org/wiki/Radioligandhttp://en.wikipedia.org/wiki/SDS-PAGEhttp://en.wikipedia.org/wiki/Spectroscopyhttp://en.wikipedia.org/wiki/Enzymehttp://en.wikipedia.org/wiki/Western_blottinghttp://en.wikipedia.org/wiki/Western_blottinghttp://en.wikipedia.org/wiki/ELISAhttp://en.wikipedia.org/wiki/Receptor_(biochemistry)http://en.wikipedia.org/wiki/Ligand_(biochemistry)http://en.wikipedia.org/wiki/Radioligandhttp://en.wikipedia.org/wiki/SDS-PAGE


7/34

measure of the amounts of different proteins in the mixture, and it is not able todistinguish between proteins with similarmolecular weight.

In order to evaluate the process of multistep purification, the amount ofthe specific protein has to be compared to the amount of total protein. The lattercan be determined by the Bradford total protein assay or by absorbance of light

at 280 nm, however some reagents used during the purification process mayinterfere with the quantification. For example, imidazole (commonly used forpurification of polyhistidine-tagged recombinant proteins) is an amino acidanalogue and at low concentrations will interfere with the bicinchoninic acid(BCA) assay for total protein quantification. Impurities in low-grade imidazolewill also absorb at 280 nm, resulting in an inaccurate reading of proteinconcentration from UV absorbance [2].

1.2.3. Sample preparation

The methods used in protein purification, can roughly be divided intoanalytical and preparative methods. The distinction is not exact, but thedeciding factor is the amount of protein, that can practically be purified with thatmethod. Analytical methods aim to detect and identify a protein in a mixture,where as preparative methods aim to produce large quantities of the protein forother purposes, such as structural biology or industrial use. In general, thepreparative methods can be used in analytical applications, but not the otherway around.

1.2.3.1. Extraction

Depending on the source, the protein has to be brought into solution bybreaking the tissue or cells containing it. There are several methods to achievethis: Repeated freezing and thawing, sonication, homogenization by highpressure or permeabilization by organic solvents.

The method of choice depends on how fragile the protein is and howsturdy the cells are. After this extraction process soluble proteins will be in thesolvent, and can be separated from cell membranes, DNA etc. bycentrifugation. The extraction process also extracts proteases, which will startdigesting the proteins in the solution. If the protein is sensitive to proteolysis, itis usually desirable to proceed quickly, and keep the extract cooled, to slowdown proteolysis.

1.2.3.2. Precipitation and differential solubilization

In bulk protein purification, a common first step to isolate proteins isprecipitation with ammonium sulphate (NH4)2SO4. This is performed by addingincreasing amounts of ammonium sulphate and collecting the different fractions

of precipitate protein. One advantage of this method is that it can be performedinexpensively with very large volumes.

7
http://en.wikipedia.org/wiki/Molecular_weighthttp://en.wikipedia.org/wiki/Bradford_protein_assayhttp://en.wikipedia.org/wiki/Meterhttp://en.wikipedia.org/wiki/Imidazolehttp://en.wikipedia.org/wiki/Structural_biologyhttp://en.wikipedia.org/wiki/Sonicationhttp://en.wikipedia.org/wiki/Homogenizationhttp://en.wikipedia.org/wiki/DNAhttp://en.wikipedia.org/wiki/Proteaseshttp://en.wikipedia.org/wiki/Precipitation_(chemistry)http://en.wikipedia.org/wiki/Ammonium_sulphatehttp://en.wikipedia.org/wiki/Molecular_weighthttp://en.wikipedia.org/wiki/Bradford_protein_assayhttp://en.wikipedia.org/wiki/Meterhttp://en.wikipedia.org/wiki/Imidazolehttp://en.wikipedia.org/wiki/Structural_biologyhttp://en.wikipedia.org/wiki/Sonicationhttp://en.wikipedia.org/wiki/Homogenizationhttp://en.wikipedia.org/wiki/DNAhttp://en.wikipedia.org/wiki/Proteaseshttp://en.wikipedia.org/wiki/Precipitation_(chemistry)http://en.wikipedia.org/wiki/Ammonium_sulphate


8/34

The first proteins to be purified are water-soluble proteins. Purification ofintegral membrane proteins requires disruption of the cell membrane in order toisolate any one particular protein from others that are in the same membranecompartment. Sometimes a particular membrane fraction can be isolated first,such as isolating mitochondria from cells before purifying a protein located in a

mitochondrial membrane. A detergent such as sodium dodecyl sulfate (SDS)can be used to dissolve cell membranes and keep membrane proteins insolution during purification; however, because SDS causes denaturation, milderdetergents such as Triton X-100 orCHAPS can be used to retain the protein'snative conformation during complete purification.

1.2.3.3. Ultracentrifugation

Centrifugation is a process that uses centrifugal force to separatemixtures of particles of varying masses or densities suspended in a liquid.When a vessel (typically a tube or bottle) containing a mixture of proteins orother particulate matter, such as bacterial cells, is rotated at high speeds, theangular momentum yields an outward force to each particle that is proportionalto its mass. The tendency of a given particle to move through the liquid becauseof this force is offset by the resistance the liquid exerts on the particle. The neteffect of "spinning" the sample in a centrifuge is that massive, small, and denseparticles move outward faster than less massive particles or particles with more"drag" in the liquid. When suspensions of particles are "spun" in a centrifuge, a

"pellet" may form at the bottom of the vessel that is enriched for the mostmassive particles with low drag in the liquid. The remaining, non-compactedparticles still remaining mostly in the liquid are called the "supernatant" and canbe removed from the vessel to separate the supernatant from the pellet.

The rate of centrifugation is specified by the angular acceleration appliedto the sample, typically measured in comparison to the g. If samples arecentrifuged long enough, the particles in the vessel will reach equilibriumwherein the particles accumulate specifically at a point in the vessel where theirbuoyant density is balanced with centrifugal force. Such an "equilibrium"centrifugation can allow extensive purification of a given particle [2].

1.2.4. Chromatographic methods

Usually a protein purification protocol contains one or morechromatographic steps. The basic procedure in chromatography is to flow thesolution containing the protein through a column packed with various materials.Different proteins interact differently with the column material, and can thus beseparated by the time required to pass the column, or the conditions required toelute the protein from the column. Usually proteins are detected as they arecoming off the column by their absorbance at 280 nm [2].Many different chromatographic methods exist:

8
http://en.wikipedia.org/wiki/Membrane_proteinhttp://en.wikipedia.org/wiki/Cell_membranehttp://en.wikipedia.org/wiki/Mitochondriahttp://en.wikipedia.org/wiki/Detergenthttp://en.wikipedia.org/wiki/Sodium_dodecyl_sulfatehttp://en.wikipedia.org/wiki/Denaturation_(biochemistry)http://en.wikipedia.org/wiki/Triton_X-100http://en.wikipedia.org/wiki/CHAPS_detergenthttp://en.wikipedia.org/wiki/Centrifugal_forcehttp://en.wikipedia.org/wiki/Accelerationhttp://en.wikipedia.org/wiki/G-forcehttp://en.wikipedia.org/wiki/Membrane_proteinhttp://en.wikipedia.org/wiki/Cell_membranehttp://en.wikipedia.org/wiki/Mitochondriahttp://en.wikipedia.org/wiki/Detergenthttp://en.wikipedia.org/wiki/Sodium_dodecyl_sulfatehttp://en.wikipedia.org/wiki/Denaturation_(biochemistry)http://en.wikipedia.org/wiki/Triton_X-100http://en.wikipedia.org/wiki/CHAPS_detergenthttp://en.wikipedia.org/wiki/Centrifugal_forcehttp://en.wikipedia.org/wiki/Accelerationhttp://en.wikipedia.org/wiki/G-force


9/34

1.2.4.1. Size exclusion chromatography

Size exclusion chromatography (SEC) is also known as gel filtration(GF). GF separates proteins with differences in molecular size. Column for gel

filtration is filled by porous matrix. The principle is that smaller molecules haveto traverse a larger volume in a porous matrix. Unlike ion exchangechromatography or affinity chromatography molecules does not bind tochromatography medium so buffer composition does not directly affectresolution. Samples are eluted isocratically (single buffer, no gradient) [3].

Figure 2: Gel filtration - Principle of separation and elution [3].

In the context of protein purification, the eluant is usually pooled indifferent test tubes. All test tubes containing no measurable trace of the proteinto purify are discarded. The remaining solution is thus made of the protein to

purify and any other similarly-sized proteins.Besides its usage in Three phase purification strategy this method can be usedfor sample conditioning desalting of sample or changing sample buffer [1].

1.2.4.2. Ion exchange chromatography

Ion exchange chromatography (IEX) separates compounds according tothe nature and degree of their ionic charge. The column to be used is selectedaccording to its type and strength of charge. Anion exchange resins have a

positive charge and are used to retain and separate negatively chargedcompounds, while cation exchange resins have a negative charge and are usedto separate positively charged molecules [4].

Before the separation begins a buffer is pumped through the column toequilibrate the opposing charged ions. Upon injection of the sample, solutemolecules will exchange with the buffer ions as each competes for the bindingsites on the resin. The length of retention for each solute depends upon thestrength of its charge. The most weakly charged compounds will elute first,followed by those with successively stronger charges. Because of the nature ofthe separating mechanism, pH, buffer type, buffer concentration, andtemperature all play important roles in controlling the separation [2].

9


10/34

Figure 3: Separation of charged proteins and its elution by IEX [4].

Ion exchange chromatography is a very powerful tool for use in proteinpurification and is frequently used in both analytical and preparative separations[4].

1.2.4.3. Affinity chromatography

Affinity Chromatography (AC) is a separation technique based uponmolecular conformation, which frequently utilizes application specific resins.These resins have ligands attached to their surfaces which are specific for thecompounds to be separated. Most frequently, these ligands function in a fashionsimilar to that of antibody-antigen interactions. This "lock and key" fit betweenthe ligand and its target compound makes it highly specific, frequently

generating a single peak, while all else in the sample is unretained [5].

Figure 4: Principle of affinity chromatography. This type of chromatography uses high specificbound principle lock and key [5].

Many membrane proteins are glycoproteins and can be purified by lectinaffinity chromatography. Detergent-solubilized proteins can be allowed to bindto a chromatography resin that has been modified to have a covalently attachedlectin. Proteins that do not bind to the lectin are washed away and thenspecifically bound glycoproteins can be eluted by adding a high concentration ofa sugar that competes with the bound glycoproteins at the lectin binding site.Some lectins have high affinity binding to oligosaccharides of glycoproteins that

is hard to compete with sugars, and bound glycoproteins need to be releasedby denaturing the lectin [2].

10
http://en.wikipedia.org/wiki/Glycoproteinhttp://en.wikipedia.org/wiki/Lectinhttp://en.wikipedia.org/wiki/Oligosaccharideshttp://en.wikipedia.org/wiki/Glycoproteinhttp://en.wikipedia.org/wiki/Lectinhttp://en.wikipedia.org/wiki/Oligosaccharides


11/34

1.2.4.4. Hydrophobic interaction chromatography

HIC separates proteins with differences in hydrophobicity. The technique

is ideal for the capture or intermediate steps in a purification. The separation isbased on the reversible interaction between a protein and the hydrophobicsurface of a chromatographic medium. This interaction is enhanced by highionic strength buffer which makes HIC an ideal 'next step' after precipitation withamonium sulphate or elution in high salt during IEX. Samples in high ionicstrength solution (e.g. 1.5 M ammonium sulphate) bind as they are loaded ontoa column. Conditions are then altered so that the bound substances are eluteddifferentially. Elution is usually performed by decreases in salt concentration [6].

Figure 5: Principle of hydrophobic interaction chromatography [6].

There are many types of HIC ligands (octyl sepharose, butyl sepharose,fenyl sepharose etc.). Very hydrophobic proteins bind tightly to veryhydrophobic ligands and may require extreme elution conditions, e.g. chaotropicagents or detergents, for the target protein or contaminants.

For any chromatographic separation each different technique will offerdifferent performance with respect to recovery, resolution, speed and capacity.A technice can be optimised to focus on one of these parameters, for exampleresolution. Also it is necessary to choose logical combinations of purification

techniques based on the main benefits of the technique and the condition of thesample at the beginning or end of each step [1]. Next figure shows suitability ofparticular chromatographic techniques for Three phase purification strategy.

11


12/34

Figure 6: Suitability of purification techniques for Three phase purification strategy [1].

1.2.4.5. HPLC

High-performance liquid chromatography (HPLC) is a form ofliquidchromatography to separate compounds that are dissolved in solution. HPLCinstruments consist of a reservoir of mobile phase, a pump, an injector, aseparation column, and a detector. Compounds are separated by injecting aplug of the sample mixture onto the column. The different components in themixture pass through the column at different rates due to differences in their

partitioning behaviour between the mobile liquid phase and the stationaryphase.Solvents must be degassed to eliminate formation of bubbles. The

pumps provide a steady high pressure with no pulsating, and can beprogrammed to vary the composition of the solvent during the course of theseparation. Detectors rely on a change in refractive index, UV-VIS absorption,or fluorescence after excitation with a suitable wavelength. [7].

1.2.5. Concentration of the purified protein

A selectively permeable membrane can be mounted in a centrifuge tube.The buffer is forced through the membrane by centrifugation, leaving the proteinin the upper chamber. At the end of a protein purification, the protein often hasto be concentrated. Different methods exist.

Lyophilization

If the solution doesn't contain any other soluble component than theprotein in question the protein can be lyophilized (dried). This is commonly doneafter an HPLC run. This simply removes all volatile component leaving theproteins behind.

Ultrafiltration

12
http://elchem.kaist.ac.kr/vt/chem-ed/sep/lc/lc.htmhttp://elchem.kaist.ac.kr/vt/chem-ed/sep/lc/lc.htmhttp://elchem.kaist.ac.kr/vt/chem-ed/sep/extract/partitn.htmhttp://en.wikipedia.org/wiki/Freeze_dryinghttp://en.wikipedia.org/wiki/Lyophilizationhttp://en.wikipedia.org/wiki/Ultrafiltrationhttp://elchem.kaist.ac.kr/vt/chem-ed/sep/lc/lc.htmhttp://elchem.kaist.ac.kr/vt/chem-ed/sep/lc/lc.htmhttp://elchem.kaist.ac.kr/vt/chem-ed/sep/extract/partitn.htmhttp://en.wikipedia.org/wiki/Freeze_dryinghttp://en.wikipedia.org/wiki/Lyophilizationhttp://en.wikipedia.org/wiki/Ultrafiltration


13/34

Ultrafiltration concentrates a protein solution using selective permeablemembranes. The function of the membrane is to let the water and smallmolecules pass through while retaining the protein. The solution is forcedagainst the membrane by mechanical pump or gas pressure or centrifugation

[2].

1.2.6. Electrophoresis

Denaturing-Condition Electrophoresis

Gel electrophoresis is a common laboratory technique that can be useboth as preparative and analytical method. The principle of electrophoresisrelies on the movement of a charged ion in an electric field. In practice, the

proteins are denatured in a solution containing a detergent (SDS). In theseconditions, the proteins are unfolded and coated with negatively chargeddetergent molecules. The proteins in SDS-PAGE are separated on the solebasis of their size.

In analytical methods, the protein migrate as bands based on size. Eachband can be detected using stains such as Coomassie blue dye orsilver stain.Preparative methods to purify large amounts of protein, require the extraction ofthe protein from the electrophoretic gel. This extraction may involve excision ofthe gel containing a band, or eluting the band directly off the gel as it runs offthe end of the gel.

In the context of a purification strategy, denaturing conditionelectrophoresis provides an improved resolution over size exclusionchromatography, but does not scale to large quantity of proteins in a sample aswell as the late chromatography columns.

Non-Denaturing-Condition Electrophoresis

Non- denaturing (also named native) electrophoresis is based onto sameprinciple (movement of charged ions in electric filed) as denaturingelectrophoresis. In contrast to denaturing electrophoresis does not use SDS or

another denaturing agent and so does not take place denaturation of proteinsand proteins retain its properties which it is possible to use in its detection [2].

1.3. Metabolism of carbonyl groups

Molecular biological and biochemical studies have established the criticalimportance of carbonyl metabolizing enzymes, both in endogenous and

xenobiotic phase I metabolism.

13
http://en.wikipedia.org/wiki/Gel_electrophoresishttp://en.wikipedia.org/wiki/Electrophoresishttp://en.wikipedia.org/wiki/Sodium_dodecyl_sulfatehttp://en.wikipedia.org/wiki/SDS-PAGEhttp://en.wikipedia.org/wiki/Coomassiehttp://en.wikipedia.org/wiki/Staining_(biology)#Silver_staininghttp://en.wikipedia.org/wiki/Chromatography#Column_chromatographyhttp://en.wikipedia.org/wiki/Gel_electrophoresishttp://en.wikipedia.org/wiki/Electrophoresishttp://en.wikipedia.org/wiki/Sodium_dodecyl_sulfatehttp://en.wikipedia.org/wiki/SDS-PAGEhttp://en.wikipedia.org/wiki/Coomassiehttp://en.wikipedia.org/wiki/Staining_(biology)#Silver_staininghttp://en.wikipedia.org/wiki/Chromatography#Column_chromatography


14/34

Aldehydes are mainly converted through oxidation to the respectivecarboxyl acids or through reductive processes to the primary alcoholcompounds. Due to their chemical reactivity aldehydes are good electrophilesand can interact with cellular nucleophilic centers in nucleic acids or proteins.Most ketones, on the other hand, are less reactive and can be interconverted by

ketone reductases or alcohol dehydrogenases to the respective secondaryhydroxyl metabolites.Reductive quinone metabolism attracts special toxicological interest

since stepwise reduction of quinone compounds leads to semiquinone radicalsthat can be either reduced to hydroquinones or reconverted to quinones,involving molecular oxygen, giving rise to reactive oxygen species, and leadingto cell damage. The chemical relationships, the major pathways of carbonylgroup containing compounds, the enzyme systems and their structural andfunctional characteristics are summarized in Fig. 7.

Figure 7: The major metabolic conversions of carbonyl group containing compounds. ALDH aldehyde dehydrogenases, MDR medium chain dehydrogenase/reductase, SDR short chaindehydrogenase/reductase., AKR aldo-keto reductase, QR quinine reductase [8].

1.3.1. Carbonyl reducing enzymes

Carbonyl reduction is a significant step in the biotransformation of many

aliphatic, alicyclic and aromatic xenobiotic carbonyl compounds.Pharmacological agents include, e.g. warfarin, fenofibrate, oxisuran, ethacrynic

14


15/34

acid, and a variety of toxicologically important compounds such as aflatoxin B1or the nitrosamine NNK, which are metabolized via carbonyl reduction. Themetabolic consequence is either activation or inactivation, depending on thecompound. However, in most cases the formation of a hydroxyl group rendersthe substance more hydrophilic and provides a phase I reaction product that

can be conjugated, e.g. via glucuronidation or sulfation, thus facilitatingexcretion. The main cytosolic carbonyl reducing enzymes in mammals arecarbonyl reductase (EC 1.1.1.184), aldehyde reductase (EC 1.1.1.2), aldosereductase (EC 1.1.1.21) and dihydrodiol dehydrogenases. They arecharacterized by broad and overlapping substrate specificities and, besidestheir participation in xenobiotic phase I reactions, are also involved inendogenous steroid hormone, bile acid and arachidonic acid metabolism,catalyzing OH- dehydrogenase/keto-reductase reactions. This dual specificity isalso characteristic for microsomal carbonyl reducing enzymes like 11-hydroxysteroid dehydrogenase type I (11-HSD-1), which has been shown tobe involved in important detoxification reactions, e.g. as NNK reductase.

Quinone reductase activities have been reported for several carbonylreducing enzymes, implicating a role in oxidative stress (c.f. below). Furtherimplications derive from dihydrodiol dehydrogenase activity towards trans-dihydrodiols of activated polycyclic aromatic hydrocarbons. Although earlyreports suggest reduction of the mutagenic potential of aromatic hydrocarbonsthe resulting o-quinone products of this reaction give rise to reactive oxygenspecies and are involved in, e.g. naphthalene-induced cataracts or formation ofgenotoxic benzo(a)pyrene metabolites[8].

Figure 8: Ribbon presentation of monomeric subunits of enzyme families involved in metabolismof carbonyl groups [8].

At present most carbonyl reducing enzymes characterized are groupedinto two distinct protein superfamilies, the aldoketo reductase (AKR) and the

short-chain dehydrogenase/reductase (SDR) superfamilies [8].Name Function Coenzym Size Structure Mechanis Active

15


16/34

e m site

AKR

Aldo-ketoreductases

Oxidoreduction of endogenousandxenobiotic

carbonyls,alcohols andC_C doublebonds

NADP(H) About 320residues

(a:b)8 (TIM)Barrel; noRossmann-fold

nucleotidedomain;

Acid-basemechanismwith Tyr ascatalyticacid:base,

ordered BiBi

Tyr, Asp,Lys, His,(Glu)

SDR

Short-chaindehydrogenases/reductases

Oxidoreduction of endogenousandxenobioticcompounds;lyases,epimerases

NAD(P)(H) About 270residues

a:b-Fold,one-domainRossmann-foldnucleotidebinding site

Acid-basemechanismwith Tyr ascatalyticacid:base,ordered BiBi

Tyr, Lys, Ser

Figure 8: Characteristics of enzyme superfamilies involved in carbonyl reduction [8].

1.3.1.1. Aldoketo reductase superfamily (AKR)

The AKRs are enzyme superfamily of NAD(P)H- dependentoxidoreductases. There are found in vertebrates, invertebrates, plant, protozoaetc. It has been described more than 100 members of AKR superfamily so far,12 of them are human AKRs. In last year It was established nomenclaturesystem for AKR superfamily which divides members to families and subfamiliesaccording to its amino acid sequences (for example AKR1C3 is enzyme whichbelongs to family 1 and subfamily C). [9,10] The well-known enzyme from thissuperfamily are aldose reductase, aldehyde reductase and enzymes from

AKR1C family.

AKRs are cytosolic, monomeric (/)8-barrel proteins, about 320 aminoacids in length, which use NAD(P)(H) to metabolize a range of substrates. Thecosubstrate binding is not accomplished by a Rossmann-fold structure, theNADP(H) molecule is bound at the bottom of the barrel. Conserved residueswhich are catalytically important in the OH-dehydrogenase/carbonyl reductasereactions are Tyr, Asp, Lys, and His. Present data suggest that the reactionmechanism is a general Tyr-based acid-base catalytic mechanism, facilitated bythe conserved Lys, lowering the pKa of the tyrosine. A change in reactionmechanism is observed with the 3-oxo-5-steroid 4-dehydrogenase, catalyzing

the reduction of the steroid C---C double bond, where His is replaced by Glu,indicating a carbonium ion intermediate stabilized by this residue, followed by aMarkovnikov addition. Interestingly, the conserved Tyr residue in rat 3 -hydroxysteroid dehydrogenase/dihydrodiol dehydrogenase (3 -HSD/DHD;AKR1C9) is not essential in quinone reductase activity of quinones derived frompolyaromatic hydrocarbons, suggesting a different reaction mechanism withthese compounds, although binding to the same active site [10].

1.3.1.2. Short-chain dehydrogenases/reductases (SDR)

16


17/34

SDR constitute large, functionally heterogenous protein family, presentlycontains more than 3000 members. The enzymes included in SDR span severalclasses from oxidoreductases and lyases to isomerases, with oxidoreductasesforming the majority [11]. Cytosolic carbonyl reductase (CR) and microsomal11-HSD-1 are the two best studied xenobiotic carbonyl reducing enzymes of

the SDR family. Most of the SDR structures known are soluble proteins with amonomer length of about 270 amino acid residues. However, considerablevariation occurs in membrane-bound or multi-enzyme structures. Mono- andoligomeric forms are described. The active site comprises a catalytic triad ofhighly but not strictly conserved Ser-Tyr-Lys residues. Sequence comparisons,biochemical and structural studies suggested a Tyr based acidbasemechanism, facilitated by a conserved Lys residue, lowering the Tyr pKa, aone-domain / folding and a Rossmann-fold as nucleotide cosubstrate bindingsite [12].

Figure 9: Reaction mechanism of short-chain dehydrogenases involving the conserved Tyr andLys residues. Residue numbers refer to 3d20B-hydroxysteroid dehydrogenase [12].

Although hydride transfer is achieved from different positions (4 pro-S,SDR) and (4 pro-R, AKR) of the nicotinamide, the highly similar reactionmechanisms of the AKR and SDR families represent an example of convergent

evolution of two distinct protein families, since the corresponding catalyticallyactive site residues can be nearly superimposed in the 3D structuresdetermined (c.f. 10 B) [8].

17
http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6TCN-40315SH-9&_user=1490772&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000053052&_version=1&_urlVersion=0&_userid=1490772&md5=43ca10bcdd8320b16f33a2fab2a9786f#fig2%23fig2http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6TCN-40315SH-9&_user=1490772&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000053052&_version=1&_urlVersion=0&_userid=1490772&md5=43ca10bcdd8320b16f33a2fab2a9786f#fig2%23fig2


18/34

Figure 11: Human carbonyl reductases which participate in metabolism of xenobiotics, here arenoted its endogenous substrates [13].

It is now well established that the enzyme systems involved in carbonylmetabolism constitute an important determinant in physiological andtoxicological processes. The complexity of the whole system is recognized and

further knockout and gene transfer studies besides classical biochemicalapproaches are likely to reveal further details. Recent structure determinationsprovided a rational basis for our understanding of underlying mechanisticreaction principles, and ongoing projects will advance drug development [8].

Today is well-known that several carbonyl reductases participate inmetabolism of carbonyl xenobiotics as it is shown in Figure 12. From thisknowledge is clear that enzymes are not strict divided to groups ofbiotransformation enzymes and enzymes which participate in endogenousmetabolism. These two groups are blended together. Some other enzymes arestill awaiting that its activity toward xenobiotics substrate can be discoverybesides its eobiotics substrate. It is probably a question of time and growing

interest of scientists about this area.

18


19/34

Figure 12: Human carbonyl reductases and its xenobiotics substrates [13].

1.4. Oracin

Oracin, 6-[2-(2-hydroxyethyl)aminoethyl]-5,11-dioxo-5,6-dihydro-11H-indeno[1,2-c] isoquinoline, is a promising potential cytostatic drug for oral use,which is already in phase II of clinical trials. From its chemical structure, a DNAintercalation mode of action can be inferred, similar to that of anti-tumorantibiotics from the anthracycline group (synthetic anthraquinones). However,

the wide spectrum of tumors that are sensitive to oracin does not only resultfrom this single mechanism of action, which manifests itself by an inhibition ofDNA and RNA synthesis, followed by a decrease in protein content in tumorcells. Several other mechanisms affecting tumor cell growth have beendemonstrated. They involve the inhibition of topoisomerase II which wasisolated from nuclei of Ehrlich ascites carcinoma cells, the stimulation ofaerobic consumption of glucose and, to a lesser extent, of formation of lactatein tumor cells, as well as induction of apoptosis.

Carbonyl group of oracin is pro-chiral centre, it is metabolised inreductive manner to two enatiomers of 11-dihydrooracin (DHO) [14].

19


20/34

Figure 13: Metabolic conversion of the pro-chiral molecule oracin to(+)-DHO and ()-DHO enantiomers [14].

2. Methods

20


21/34

2.1 Preparation of microsomal fraction

Microsomes are small balls which are originated form membrane ofendoplasmatic reticulums rupture. Microsomal fraction is prepared from humanliver tissue. First is necessary to disrupt liver cells by homogenization. Then thehomogenized cells and another fraction are centrifuged at different rates.Prepared microsomes are stored in freezer at -80C. It is necessary to work incooling room and centrifuge at 0C. I didnt participate in this step, because weused prepared microsomes from freezer.

2.2 Solubilization of microsomes

During solubilization of microsomes, the microsomal membranes openand stay in solution. In this step are used different types of detergents.

First it was necessary place the microsomes on the ice and let themdefrost (about 1 hour). Then we removed the amounts of 5 and 20ul to theEppendorfs and placed them to the freezer for next experiments. After it weplaced the microsomes to the beaker and added the same amount of thesolubilization buffer (10 mM NaH2PO4, 1 mM EDTA, 1 M NaCl, 40 % (w/v)glycerol and 0,4 % (w/v) Lubrol PX, pH 7,4). Then it was required to place it onthe ice and shack it for 45 minutes at laboratory temperature. Subsequently wepipetted the microsomes to the Ultra tubes (special tubes for centrifugation),balanced the amount and ultracentrifugated for 60 minutes/4C,32000turns/min. Then we placed the supernatant to the beaker and added the

natrium cholate in the concentration of 0,4% (w/v). After dissolving the natriumcholate we removed the amounts of 50 and 100ul to the Eppendorfs and placethem to the freezer.

2.3 Desalting of microsomes

This step serves to remove salt from protein sample and for this purposewe used gel filtration. This is essential when salt in sample interfere with nextstep, for example, when we used separation on Q Sepharose that is ion-exchange chromatography and in the presence of salt it is not possible bindprotein in column so it is required to remove it. However salt does not interferewith separation on hydrophobic interaction chromatography.

We used solubilized microsomes to desalting. We used apparatus ktaPurifier (Amersham Biosciences) that is low-pressure chromatograph which isused for purification of proteins. First it was required insert in Akta suitablecolumn, in which for desalting of microsomes was used Desalting Hi Trap 5 mlcolumn. First we had to wash the apparatus column with 20 % ethanol (at least5 volume of column), water (similar volume) and appropriate buffer (similarvolume). Separation preceded at flow rate 2 ml/min and we loaded onto column

repeatedly with 1ml solubilized microsomes. We monitored separation by UVdetection at 280 nm and conductometry. Desalting of microsomes was manual

21


22/34

work, it was required to collect only fraction with high absorbance at 280 nm(detection for proteins) and fraction with high conductivity (detection of salt) putin the waste. Desalted fraction was put together and concentrated.

2.4. Concentration of desalted microsomes

When we use gel filtration, the sample is always dissolved so it isnecessary to concentrate it. One of the possibilities to concentrate the sample isultracentrifugation. Its principle is described in introduction part.

We used special tubes for ultracentrifugation Amicon Ultra K 10K andcentrifugation was performed at 4C, 4000 g. The time depended on start andrequired end volume. Amicon tubes contain special cellulose membrane that ispermeable only for molecules small than 10 kDa, so proteins stay in space uponmembrane.

2.5. Separation on Q Sepharose

Q sepharose is an ion-exchange chromatography, named strong anionexchanger. Matrix in column is sepharose. Charged groups are covalentlybound to matrix, in case of Q sepharose charged group are quaternaryammonium groups. These groups are positively charged so they interact withnegatively charged groups from sample. Negatively charged proteins bindreversible to the gel. Unbound substances (positively charged and withoutcharge) are washed out from column. Bounded proteins are removed fromcolumn by gradient of salt [4].

Used buffers:Buffer A: Buffer B:20 mM Tris HCl Buffer A + 1M NaCl10 % (w/v) glycerol pH 8,00,2 mM merkaptoethanolpH 8,0 (adjust with HCl)

Separation on Q Sepharose was used on kta apparatus with connected

HiTrap Q FF 1ml column. First it was required to wash column with 20% ethanol(at least 5 volume of column), water (similar buffer), buffer B (to maximumconductivity), buffer A (to zero conductivity). We loaded 1 ml of concentrateddesalted microsomes and used prepared method on kta purifier. The boundproteins were washed from column by using gradient of salt (buffer B). Then wecollected fraction and put them to freezer at 80C. From each fraction we took10 ul for incubation and 150 ul for another experiments.

2.6. Incubation of enzyme fractions with oracin

22


23/34

Incubation of enzyme fraction with oracin is used to determinate thereducing activity in fraction. The enzymes in fraction metabolise oracin todihydrooracin and from increases of metabolite DHO it is possible to determinereducing activity.

Solutions:0,1 M phosphate buffer, pH 7,40,1M MgCl25 mM oracin (1,85 mg oracin/1ml H20)25% NH3Ethyl acetateNADPH-regeneration system:

0,8 mM NADP+ .4 mg6mM glucose-6-phosphate. 12 mgGlucose-6-P-dehydrogenase. 10 ul (add before using)3mM MgCl2 200 ul 0,1M MgCl2

0,1M phosphate buffer, pH 7,4... 200 ul

We defrosted 10 ul of enzyme fractions from freezer on ice and preparedreaction mix in eppendorfs with enzyme fraction (overall volume 100 ul)

Enzyme fraction 10 ulNADPH regeneration system 20 ul0,1 M phosphate buffer 60 ul

5mM oracin 10 ulWe had different volume of enzyme fraction - 5 ul from microsomes, 10 ul

from fractions so it was necessary to increase the volume of 0,1M phosphatebuffer to got 100 ul overall volume of reaction mix. We inserted the reaction mixwithout oracin in incubator for 5 min at 37C (preincubation). The reaction wasstarted in each eppendorfs with adding of 10 ul oracin (its necessary to start thereaction exactly after 30 s or exactly after 1min), shock the mix on shaker andput it in incubator. Time of incubation is accurately 30 minute

The reaction was stopped with adding of 40 ul 25%NH3 and then 300ulethyl acetate and then it was shocked it on shaker for 10 s and putted on ice

(its necessary to stop the reaction in each eppendorfs in the same sequence asyou start it). Eppendorfs with ended reaction were centrifuged for 2 minutes,13000 turns/sec. We removed upper layer to new eppendorf and evaporatedsamples at 45C in eppendorf concentrator (open eppendorfs). It was possibleevaporated samples store in fridge (for later analyse) or dissolve in 250 ul ofMobile phase (prepared for HLPC analysis). For better dissolving we putsamples to ultrasonic pool.

We pipetted samples from eppendorfs to glass inserts and put it in vialsand closed it. Samples were prepared for HPLC analysis.

23


24/34

2.7. HLPC method determination of totalamount of dihydrooracin (DHO) in fraction

This method is used for determination of reducing activity in samples

after incubation enzyme fractions with oracin. The main reductive metabolite oforacin is dihydrooracin (DHO) so from its amount is possible determinatereducing activity of any sample.

Column: BDS Hypersil C18 (250x4 mm)Mobile Phase (MF): 10 mM hexansulfonate buffer with 0,1M trietylamine(7,1 ml/l) pH 3,27 (adjust with H3PO4):ACN in the rate of 75:25Flow rate: 1,5 ml/minPressure: about 200 barTemperature: 25CDetection: Fluorescence detektor Ex = 340 nm, Em = 418 nm

First we switched on all components of HPLC (Agilent) and computerwith special programme instrument online. We washed the HPLC system withcolumn BDS Hypersil C18 with methanol for about one hour (flow rate 1ml/min),then with water for about one hour (flow rate 1 ml/min) and with mobile phase (itis necessary to tighten the bottle with mobile phase with parafilm because ACNis very volatile), first about 20 minute to waste. In this time the MF removedwater from HPLC system and then there was only MF. Than we reused the MF(put the line from waste to bottle with MF) and switched on the fluorescentdetector and set the flow rate to 1,5 ml/min. After achievement the balance insystem (more 30 minutes), first we analyzed 10 ul of standard of DHO(10 ug/ml). Retention time of DHO was 2,7 min. Analysis of standard wouldreveal possible mistakes in HPLC system. Because analysis of standard wasOK, we started with analysis of sample. The analysis of each sample lasted 22minutes.

2.8. Chiral HPLC analysis determination ofenantiomers of DHO

Chiral HPLC analysis serves for determination of enantiomers of DHO.This determination is very important because whole project is based on differentstereospecifity of distinct enzymes. Human purified 11-HSD1 reduces Oracinin stereospecific manner, preferably is formed enantiomer (-)-DHO (76%).Because whole liver microsomes reduced oracin to DHO in the rate of (-)-DHOand (+)-DHO 60:40, we anticipate that the new carbonyl reductase metabolizeoracin preferably to (+)-DHO.

Column: Chiracel OD-R OCE (A-1035), 250 x 4,6 mMobile phase: perchlorate buffer pH 3,0 : ACN = 69:31 (v/v)Flow rate: 0,5 ml/min

Temperature: 25 CPressure: 4,1 MPa (max. 5 MPa)

24


25/34

Detection: fluorescence detector Ex = 340 nm, Em = 418 nm

Procedure was similar to determination of total amount of HPLC. Thiscolumn is only sensitive to high pressure therefore is necessary to use lowerflow rate.

3. Results and discussion

25


26/34

Human liver whole microsomes in volume of 8,8 ml were solubilized bysolubilization buffer, centrifuged and then natrium cholate was added. Volumeof solubilized microsomes was 16,5 ml. Then we desalted the solubilizedmicrosomes by gel filtration on kta purifier. Total volume of desalted

microsomes was 42,6 ml and after concentration only 2,5 ml. 1 ml ofconcentrated desalted microsomes was applied to Q Sepharose column. Eachexperiment was performed in duplicate, in order to obtain 2 chromatograms(runs).

Figure 14: Separation of concentrated desalted microsomes on Q sepharose. Run 1.

26


27/34

Figure 15: Separation of concentrated desalted microsomes on Q sepharose. Run 2.

Each fraction from these two separation runs were incubated with oracin

to found the fraction with most reductive active fraction. Not only the fractionsfrom Q sepharose were incubated, but also from microsomes, solubilized,desalted and concentrated desalted microsomes. The reducing activity isexpressed as total amount of DHO in the fraction in ng.

sample

incubatedvolume(ul)

load(l)

dilution(l)

DHO inload(ng)

DHO in250l(ng)

volume offraction(l)

total DHOin fraction(ng)

% inactivity

Microsomes 1

100 250

20,133

50,333

8800,000

442926,000

100,000

Solubilizedmicrosom

es 10100 250

86,125

215,313

16500,000

355265,625 80,210

Desaltedmicrosomes 10

100 250

27,941

69,853

42600,000

297571,650 67,180

Concentrateddesaltedmicrosomes 1

100 250

42,449

106,123

2500,000

265306,250 59,900

Figure 16: Results after incubation of different microsomal fraction.

27


28/34

From results in Figure 15 is evident that handling with sample broughtdecreasing in enzyme activity even if whole work was carry out on ice. It is notpossible to avoid decreasing in activity but it is necessary to minimise it.

Run 1Loaded 1ml concentrated desalted microsomes: 106122,5 ngDHO

sample

incubatedvolume(ul)

load(l)

dilution(l)

DHO inload(ng)

DHO in250l(ng)

volumeoffraction(l)


% loadedactivity

A1 10100 250 0,000 0,000

1000,000 0,000 0,000

A2 10100 250

73,921

184,803

1000,000

18480,250 17,414

A3 10

10

0 250

16,99

1

42,47

8

1000,0

00

4247,75

0 4,003

A4 10100 250 2,278 5,695

1000,000 569,500 0,537

A5 10100 250 0,910 2,275

1000,000 227,500 0,214

A6 10100 250 0,502 1,255

1000,000 125,500 0,118

A7 10100 250 0,389 0,973

1000,000 97,250 0,092

A8 10100 250 0,404 1,010

1000,000 101,000 0,095

A9 10100 250 0,302 0,755

1000,000 75,500 0,071

A10 10100 250 0,480 1,200

1000,000 120,000 0,113

A11 10

10

0 250

7,67

5

19,18

8

1000,0

00

1918,7

50 1,808

A12 10

10

0 250

15,2

82

38,20

5

1000,0

00

3820,5

00 3,600

B12 10

10

0 250

8,06

5

20,16

3

1000,0

00

2016,2

50 1,900

B11 10

10

0 250 4,098

10,24

5

1000,0

00

1024,50

0 0,965

B10 10100 250 1,867 4,668

1000,000 466,750 0,440

B9 10100 250 0,967 2,418

1000,000 241,750 0,228

B8 10100 250 0,564 1,410

1000,000 141,000 0,133

B7 10100 250 0,292 0,730

1000,000 73,000 0,069

B6 10100 250 0,314 0,785

1000,000 78,500 0,074

B5 10 100 250 0,155 0,388 1000,000 38,750 0,037

28


29/34

B4 10100 250 0,135 0,338

1000,000 33,750 0,032

B3 10100 250 0,134 0,335

1000,000 33,500 0,032

B2 10

10

0 250 0,140 0,350

1000,0

00 35,000 0,033

B1 10100 250 0,117 0,293

1000,000 29,250 0,028

C1 10100 250 0,109 0,273

1000,000 27,250 0,026

C2 10100 250 0,123 0,308

1000,000 30,750 0,029

C3 10100 250 0,096 0,240

1000,000 24,000 0,023

C4 10100 250 0,150 0,375

1000,000 37,500 0,035

C5 10 100 100 0,107 0,268 1000,000 26,750 0,025

34141,

750

32,17

2

Figure 17: Results after incubation of fraction from run 1 separation on Q Sepharose.

RUN 2Loaded 1ml concentrated desalted microsomes: 106122,5 ngDHO

sample

incubatedvolume(ul)

load(l)

dilution(l)

DHO inload(ng)

DHO in250l(ng)

volumeoffraction(l)


%loadedactivity

C6 10100 250 0 0,000

1000,000 0,000 0,000

C7 10100 250

66,243

165,608

1000,000

16560,750 17,414

C8 10100 250

17,090

42,725

1000,000

4272,500 4,003

C9 10100 250 2,447 6,118

1000,000 611,750 0,537

C10 10100 250 0,423 1,058

1000,000 105,750 0,214

C11 10100 250 0,361 0,903

1000,000 90,250 0,118

C12 10100 250 0,307 0,768

1000,000 76,750 0,092

D12 10100 250 0,282 0,705

1000,000 70,500 0,095

D11 10100 250 0,217 0,543

1000,000 54,250 0,071

29


30/34


31/34

fraction

incubatedvolume(ul)

(+)-DHOin load(ng/100ul)

(-)-DHOin load(ng/100ul)

rate(-) DHO/(+) DHO

Concentrateddesaltedmicrosomes 10 121,198 169,038 58,24:41,8

run 1

A2 10 45,984 115,203 71,5:28,5

A3 10 10,451 19,287 64,9:35,1

A11 10 7,671 1,582 19,2:80,8

A12 10 13,818 4,024 22,6:77,4

B12 10 8,026 5,733 41,8:58,2

run 2

C7 10 44,544 112,666 71,7:28,3

C8 10 14,615 26,819 64,7:35,3

D9 10 6,323 1,557 19,7:80,3

D8 10 13,502 4,377 24,5:75,5

D7 10 6,991 4,908 41,2:58,8

Figure 19: Chiral analysis of the most active fraction and flow through fraction.

From chiral analysis is evident that the most active fractions (A11, A12,D9, D8) reduce Oracin preferably to (+)-DHO enantiomer, which correspondswith the primary idea. It is necessary to separate this fraction by nativeelectrophoresis and subsequently to analyse the active bands by MS analysis toreveal which enzymes are included in the most active fraction.

31


32/34

4. Conclusion

As it was referred, the development of techniques and methods for

protein purification has been an essential pre-requisite for many of the

advancements made in biotechnology [1]. This study was conducted with the

intention to establish such a technique, which is indispensable assumption of

further research.

Purification of the carbonyl reductase is not complete. It is necessary to

analyse samples on native electrophoresis. This method and subsequent MS

analysis was done in the end of my study stay. Nevertheless, our data suggest

that in reductive active fraction was present only one enzyme, which has been

reported as an enzyme with reductive activity. Our further approach is to

confirm its activity toward oracin.

32


33/34

5. Acknowledgements

Doc. Ing. Vladimir Wsol, Ph.D., head of the Department of Biochemical

Sciences, was the supervisor of this project.

I would like to express a special thanks to the Ph.D. students, especially

to Lucka Skarydova for all the support.

33


34/34

6. References

[1] Anon (2001): Protein purification handbook. Handbook of Amersham

Biosciences.

[2] http://en.wikipedia.org/wiki/Protein_purification, Wikipedia, The freeencyclopedia.

[3] Anon (2002): Gel filtration. Principles and Methods. Handbook of AmershamBiosciences.

[4] Anon (1999): Ion exchange chromatography. Principles and methods.Handbook of Amersham Biosciences.

[5] Anon (2002): Affinity chromatography. Principles and methods. Handbook ofAmersham Biosciences.

[6] Anon (1993): Hydrophobic interaction chromatography. Principle andmethods. Handbook of Amersham Biosciences.

[7] http://elchem.kaist.ac.kr/vt/chem-ed/sep/lc/hplc.htm

[8] Oppermann, U., Maser, E., 2000. Molecular and structural aspects ofxenobiotic carbonyl metabolizing enzymes. Role of reductases and

dehydrogenases in xenobiotic phase I reaction. Toxicology. 144, 71-81.[9] Jez, J.M., Penning T.M.,2001. Aldo-keto reductase (AKR) superfaimly: anupdate. Chem-Biol Interact. 130-132, 495-525.

[10] http://www.med.upenn.edu/akr/

[11] Oppermann, U., Filling, Ch., Hult, M., Shafqat, N., Wu, X., Lindh, M.,Shafqat, J., Nordling, E., Kallberg, Y., Persson, B., Jornvall, H., 2003. Short-chain dehydrogenases/reductases (SDR): the 2002 update. Chem-Biol Interact.143-144, 247-253.

[12] Jornvall, H., Bengt, P., Krook, M., Atrian, S., Gonzlez-Duarte, R., Jeffery,J., Ghosh, D., 1995. Short-Chain Dehydrogenases/Reductases (SDR).Biochemistry. 34, (18).

[13] Matsunaga, T., Shintani S., Hara A., 2006. Multiplicity of MammalianReductases for Xenobiotic Carbonyl Compounds. Drug Metab. Pharmacokinet.21 (1), 1-18

[14] Wsl V., Szotkov, B., Sklov, L., Maser, E., 2004. The novel anticancerdrug oracin: different stereospecificity and cooperativity for carbonyl reduction

by purified human liver 11 Beta-hydroxysteroid dehydrogenase type 1.Toxicology 197 253 261
http://en.wikipedia.org/wiki/Protein_purificationhttp://www.med.upenn.edu/akr/http://en.wikipedia.org/wiki/Protein_purificationhttp://www.med.upenn.edu/akr/

Date post:	30-May-2018
Category:	Documents
Upload:	zealeus
View:	215 times
Download:	0 times

Purification of a New Human Carbonyl Reductase - Report

Documents