Vol. 42, No. 4, July 1997 BIOCHEMISTRY end MOLECULAR BIOLOGY INTERNATIONAL Pages 759-768

PROTOPORPHYRINIX POTENTIATES HORSERADISH PEROXIDASE- CATALYZED OXIDATION OF NADH:INVOLVEMENT OF ENZYME-PORPHYRIN

INTERACTION

Susmita Sil & Abhay S Chakraborti.

Department of Biophysics, Molecular Biology & Genetics, University College of Science,

92, Acharyya Prafulla Chandra Road, Calcutta - 700 009, India.

Received November 10, 1996 Received after revision April 18, 1997

SUMMARY

Protoporphyrin IX potentiates horseradish peroxidase-catalyzed hydrogen peroxide-mediated NADH oxidation, but the porphyrin cannot change the enzyme-catalyzed o-dianisidine oxidation. Spectrofluorimetric studies reveal that an interaction occurs between horseradish peroxidase and protoporphyrin IX. The interaction is predominantly hydrophobic and entropy-driven endothermic process. This interaction may influence the potentiation effect of the protoporphyrin IX on horseradish peroxidase-catalyzed NADH oxidation because the latter has a positive correlation with the extent of binding of the protein with the porphyrin.

INTRODUCTION

Porphyrin IX species such as deuteroporphyrin IX, hematoporphyrin IX,

hematoporphyrin derivative, mesoporphyrin IX and protoporphyrin IX are

photosensitizers that can be activated by visible light, yielding highly reactive

photoexcited species [1]. Their photosensitizing properties have been extensively

studied at several levels (including the clinical) [2,3]. As a result, photochemotherapy

using porphyrin species has been established as a new modality of cancer treatment

[4,5]. Besides photosensitizing properties, porphyrins also have dark effects on the

biological processes [6]. Here we have investigated the effects of protoporphyrin IX (PPIX) on horseradish peroxidase (EC 1.11.1.7, donor H20 2 oxidoreductase). PPIX is

the iron-free precursor of heme. Horseradish peroxidase (HRP), an important heme

protein and viewed as a model enzyme, catalyzes primarily the oxidation of a wide

variety of donor molecules by hydrogen peroxide. Some porphyrins have been

ABBREVIATIONS: HRP, Horseradish peroxidase; PPIX, Protoporphyrin IX; SOD, Superoxide dismutase and O:~, Superoxide radical.

Corresponding Author : A.S.Chakrabarti.

1039-9712/97/040759-10505.00/0 Copyright �9 1997 by Academic Press Australia.

759 All rights of reproduction in any form reserved.

Vol. 42, No. 4, 1997 BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL

reported to have pronounced potentiating effect on the HRP-catalyzed oxidation of

NADPH, in a non-photodynamic manner [7-9]. However, different mechanisms

have been proposed for this potentiation of HRP-catalyzed oxidation of NADPH

by porphyrins. According to Bodaness [7], the enzymatic generation of

hematoporphyrin transients, which are usually generated photodynamically may

be responsible for this potentiation, van Steveninck et al. [8,9], on the other hand,

have suggested that superoxide radical (O~) generation in the HRP/H2�9

system is prevented by the porphyrin (hematoporphyrin derivative) leading to the

potentiation of enzymatic activity. However, no study has been made on the

interaction of porphyrin with HRP. Here we report the effect of PPIX on HRP activity

as well as different binding parameters for the interaction of HRP with the porphyrin at

its different concentration ranges. We also demonstrate that the potentiation effect of

PPIX on HRP has a positive correlation with the extent of binding of the ligand with

the enzyme.

MATERIALS AND METHODS

Materials: Disodium protoporphyrin IX, horseradish peroxidase (type 1), o-dianisidine, superoxide dismutase (SOD) and NADH were obtained from Sigma Chemical Company, USA. Other chemicals were of analytical grade and purchased locally.

Preparation of PPIX solution: PPIX crystals taken in 0.15 M NaCI was stirred for an hour and centrifuged. A known amount of aliquot from the supernatant (stock solution) was diluted in final concentration of 2.7 (N) HCI. The concentration of PPIX in this

acidic solution was determined using extinction coefficient ~:4o8 nm ----" 262 mM-lcm -1 in 2.7 (N) HCI [10]. The stock solution was properly diluted with 0.15 M NaCI for enzyme assay and binding experiments. Solutions of PPIX were always freshly prepared and protected from light.

Enzyme assay: All enzymatic assays were done in a Hitachi U2000 spectrophotometer at ambient temperature with buffer in the reference cell. A typical reaction mixture contained 10 mM sodium phosphate buffer, pH 7.4, 0.05 mg/ml HRP, 0.1 mM NADH and different concentrations of PPIX as indicated. In control experiments PPIX was omited and its volume was made up with 0.15 M NaCI. The reaction was initiated by addition of 98 t~M of H202. Change in absorbance at 340 nm

was followed for 3 minutes under continuous illumination. PPIX in buffer did not exhibit any change in absorbance when illuminated similarly at 340 nm for 3 minutes. In the experiments where effect of SOD on HRP-catalyzed NADH oxidation was observed, SOD was added instead of PPIX.

For HRP-catalyzed oxidation of o-dianisidine, the reaction mixture contained 100 mM sodium phosphate buffer, pH 6.0, 83.4 ng/ml HRP, 0.005% o-dianisidine and PPIX concentrations as indicated. The reaction was initiated by adding 2.2 mM H202. Change in absorbance at 460 nm was followed for 3 minutes.

760

Vol. 42, No. 4, 1997 BIOCHEMISTRYand MOLECULAR BIOLOGY INTERNATIONAL

Binding experiments: Interactions between PPIX and HRP were assayed spectrofluorimetrically in Hitachi F3010 spectrofluorimeter (equipped with a thermostated bath) using a 1 cm pathlength quartz cuvette. Titrations of protein fluorescence were performed by adding PPIX from a concentrated stock solution to the protein. The volume increase in the cuvette was negligible. Concentration of HRP used was 0.44 I~M. Titration was done with two concentration ranges of PPIX : 0.3-1.5 ~LM and 1.5-3.0 I~M. Absorbance of the maximum concentration of porphyrin (3 ~LM) in normal saline at the excitation wavelength was less than 0.05 and the entire range used (0.3-3.0 i.tM) followed Beer's relation. Excitation was done at 285 nm with excitation bandpass 5 nm and emission bandpass 10 nm. The emission peak emerged at 340 nm. Spectra were noted after baseline correction. Different binding parameters between HRP and PPIX were measured as described in the Results and Discussion section.

RESULTS AND DISCUSSION

Porphyrins were reported to have pronounced potentiating effect on the HRP-

catalyzed oxidation of NADPH in a non-photodynamic manner [7-9]. Effects of

hematoporphyrin [7] and hematoporphyrin derivative [8,9] were mostly studied. In the

present study, PPIX was used as the porphyrin species and it was found to potentiate

very efficiently the HRP-catalyzed oxidation of NADH (Table 1). 10 p.M PPIX increased

the enzyme activity about 3.5-fold (0.113_+0.010) over the control activity

(0.032+0.003). However, it had almost no effect on HRP-catalyzed oxidation of

o-dianisidine. Steveninck et al. [8] also showed that HRP-catalyzed oxidation of

guaiacol was not potentiated by hematoporphyrin derivative.

Attempts were made to understand the non-photodynamic effect of PPIX on

HRP activity. Takayama and Nakano [11] demonstrated the generation of superoxide

anion (O~) in the course of NADH oxidation in the HRP-H202 system, van Steveninck

et al. [8] reported that O~ generation in HRP/H202/NADPH system was inhibited by

hematoporphyrin derivative causing potentiation of the enzyme activity. We also found

that SOD increased the velocity of HRP-catalyzed oxidation of NADH. For example,

SOD (45 units) could potentiate the enzyme-catalyzed NADH oxidation by about

1.7-fold (Fig.l). van Steveninck et al. [9] suggested that porphyrin anion radical derived

from hematoporphyrin derivative might be responsible for inhibition of O~ in

HRP/H202/NADPH system. However, the underlined mechanism of the potentiation of

HRP-catalyzed oxidation of NADPH/NADH by porphyrins is still not clear. Studies on

the interaction of PPIX with HRP may be important in this respect. This type of interaction may prevent O~ generation in HRP/H202/NADH system and/or directly

influence the enzyme leading to its increased activity. Here we have studied the

interaction between HRP and PPIX.

Porphyrin tX species tend to aggregate in aqueous medium [12,13]. We have

recently seen that the binding nature of PPIX with hemoglobin and myoglobin changes

761

Vol. 42, NO. 4, 1997 BIOCHEMISTRYand MOLECULAR BIOLOGY INTERNATIONAL

TABLE 1 : Effects of PPIX on HRP activity

System Rate of NADH oxidation Rate of o-dianisidine oxidation (AOD340nm/min) (AOD460nm/min)

Only HRP (Control) 0.032 + 0.003 0.812 + 0.06

HRP + 5.0 laM PPIX 0.075 + 0.003 0.799 + 0.08

HRP+ 10.0 p.M PPIX 0.113+0.010 0.785+0.04

Results are mean + SEM of 5 observations.

0 . 6

o

=o 0.4 122 0 U3 O3 < 0.3

0-2 t i t

0 60 120 180

TIME (Sec)-"

Fig.1 : Effect of SOD and PPIX on the rate of HRP-catalyzed H202-mediated NADH oxidation. Control activity (o); activity in presence of 45 units of SOD (-) and 10 ~tM PPIX (A).

with varying aggregation state of PPIX [14]. In the present study of HRP-PPIX

interaction, we used two concentration ranges of PPIX (0.3-1.5 ~tM and 1.5-3.0 /.tM)

According to Margalit et al. [12], dimerization is the dominant aggregation process in

&001-0.1!aM range of PPIX concentration and higher aggregation should not be

neglected exceeding this concentration. Therefore, a significant amount of PPIX in the

concentration range used in this study should be ~in aggregated states. The extent of

aggregation probably differs as the concentration range of PPIX is increased from

0.3-1.5 p.M to 1.5-3.0 p.M. However, an equilibrium probably exists between aggregated

and non-aggregated forms of the porphyrin within these concentration ranges.

7 6 2

Vol. 42, No. 4, 1997 BIOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL

Binding parameters were determined from the quenching of protein

(HRP) f luorescence at 340 nm with gradual addition of PPIX. The quenching data were

analyzed to determine the binding affinity constant (K) and the possible number of

binding sites (p).

F ig2 is the l inear plot of Fo/AF versus I /L t following the equat ion[15] :

Fo/AF = Fo/AFma x + Fo/AFma x, I / K . 1/L t

where AF=F o - F; F o and F represent the fluorescence intensity of HRP at 340 nm in

the absence and presence of the added PPIX concentration (Lt), respectively. AFma x is

the maximum change in f luorescence intensity. The intercept of the above plot on the

Fo/AF axis at 1/L t = 0 gives an estimate of the Fo/AFma x and the slope measures the

affinity constant, K. It was found that K value decreased as the concentration range of

PPIX increased.

The possible number of binding sites (p) were estimated from the plot of 1/(1-0) versus Lt/0 (Fig.3) following the relation [15]:

11(1-0)=K. Lt/0 - K . p . A t

where 0 is the fractional saturation of PPtX sites expressed as 0 =AF/AFma x and A t is

50

40

30

5x

20

10

it.

I I i 1 2 3

( ~u I'~1)-,, 1 L t

Fig.2 �9 Plot of Fo/AF versus l iLt, where L t is the total concentration of PPIX added to HRP

(0.44/aM). Concentration ranges of PPIX used were 0.3 - 1.5 ~tM (o) and 1.5 - 3.0 I~M (A).

763

Vol. 4 2 , No. 4 , 1 9 9 7 B IOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL

2.00

1'75

f $1-5o

1"2 5

1"00

0"75 I

0 2

o;; o / I I I _ 4 6 8

Lt e (p M)--~

Fig.3 �9 Plot of 1/(1-0) versus Lt/O where 0 is the extent of binding and L t is the concentration of PPIX. The concentration of HRP was 0.44 #M and the PPIX concentration ranges used were 0.3 - 1.5 #M (o) and 1.5 - 3.0 pM (A).

the fixed concentration of protein. The number of binding sites appeared to be

approximately 1.

It is known that PPIX binds to different proteins e.g., human serum albumin [16],

hemopexin [16], hemoglobin [14,17,18], myoglobin [14], low and high density

lipoprotein [19] etc. Our results indicate that PPIX binds also with HRP The smaller

aggregates present in low concentration range ( 0.3-115 pM ) of PPIX probably bind

with higher affinity (K=4.55+0.35x105M -1) than the larger aggregates

(K=1.96+0.35x105M -1) present in higher concentration range (1.5-3.0 ~tM) However,

the lower and higher aggregates probably bind at the same site on HRP

Experiments were also done to determine the nature of PPIX-HRP interaction.

Binding experiments were done at different molarities of NaCI ranging from 0.05-0.3 M

For both PPIX concentration ranges, the affinity constant values for HtRP-PPIX complex

did not change significantly with increased NaCI molarity indicating predominantly a

non-electrostatic interaction (data not shown).

Enthalpy and entropy changes accompanying the binding process were

estimated to have further understanding of the nature of interaction. The temperature

dependence of the binding constant was measured spectrofluorimetrically from the

emission spectra of HRP in presence of PPIX in the temperature range of 15~ to

764

Vol. 42, No. 4, 1997 BIOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL

35~ This temperature range did not have any effect on fluorescence emission spectra

of either protein or PPIX. Fig.4 shows the van't Hoff isochore plot of InK versus IO00/T

where T is the absolute temperature at which the PPIX binding to HRP was studied.

The slope of this plot measures the standard enthalpy change (AW) following the

equation [20]:

InK = - AG~ = - AH~ + AS~

where AG ~ is the Gibb's free energy change upon binding of PPIX to HRP and AS ~ is

the corresponding standard entropy change. Assuming no significant temperature

dependence'of AH ~ in the temperature range used, the value of AH ~ AS" and AG " were

estimated and tabulated (Table 2). For both concentration ranges of PPIX, protein-

porphyrin interaction appeared to be endothermic (AH ~ value positive) But AS"

assumed such a high positive value that the entropic contribution (T.AS~ in the

equation AG ~ = AH ~ - T.AS ~ ultimately enabled AG ~ to become negative making the

binding process a favourable one. Such entropy driven process is expected for binding

of ligands by primarily hydrophobic process [21]. Such hydrophobic interaction possibly

involves an intermediate collisional process that becomes enhanced with the rise in

temperature and hence the association binding constant increases with the increase of

temperature.

Experiments were done to test whether the potentiation effect of PPIX on HRP-

catalyzed NADH oxidation had any correlation with HRP-PPIX interaction. The rate of

NADH oxidation catalyzed by a fixed amount of HRP and H202 was assayed with

varying concentrations of PPIX (0-3.0 p.M). In each case the extent of binding of

0-10

f .~ 0.08

E C

o 0-06

Q 0

<~ 0-04

0.02 i i 0-15 0-25 0.35 0.45

8 - "

Fig.4 " Plot of InK versus IO00/T where K is the association constant for HRP at different concentration ranges of PPIX: 0.3-1.5 p,M (o) and 1.5-3,0 p_M (&). T is the temperature in absolute scale. Temperature range selected is 15oc to 35~

765

Vol. 42, No. 4, 1997 BIOCHEMISTRYand MOLECULAR BIOLOGY INTERNATIONAL

T A B L E 2 : T h e r m o d y n a m i c p a r a m e t e r s of H R P - P P I X i n t e r a c t i o n s

PPIX Standard Enthalpy Standard Entropy Gibb's Free Concentration change change Energy change

(~M) AH o AS o AG ~ (Kcal/mole) (Cal/deg/mole) (Kcal/mole)

0.3 - 1.5 + 1.68 + 31.47 - 7.69

1.5 - 3.0 + 1.73 + 30.47 - 7.22

Mean of 4 observations.

13"5

13"0

f " 12.5 C

12-0

11-5

Fig.5 " Rate of HRP-catalyzed PPIX against the extent of

I I I 3-2 3-3 3.4 3-5

1000 ( I/I< )-* T

NADH oxidation in presence of different concentrations of saturation e of the enzyme with respective porpnyrin

concentrations. Same amount of enzyme (0.05mg/ml) and PPIX (1.5 - 2.9 I~M) were used both in bindin.g experiments and assay of activities. 0 is expressed as AF/AFma x.

HRP with PPIX (0) was also determined from spectrofluorimetric experiments

using the relation O = AF/AFma x. The enzyme activities (AOD340nm/min) were plotted

against 0 (Fig5). The enzyme activity was found to have significant positive correlation

With the bound fraction of PPIX as determined from the correlation coefficient r = 0,9

(approx.) using the relation [22]:

r = , ~(x;:~).(y-~)

,/Z(x-2)2.Z(y-~) 2

766

Vol. 42, No. 4, 1997 BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL

where x and y stand for extent of ligand binding and extent of enzyme activity at

different PPIX concentrations, respectively. ~, y represent the mean values

Interaction between HRP and potentiation of HRP-catalyzed oxidation of NADH

by PPIX have been found to be correlated (Fig.5). The potentiation effect of PPIX on the enzyme activity may be due to inhibition of 0 2 generation in HRP/H202/NADH

system. The latter effect (generation or inhibition of O~ ) may, in turn, be caused due to

interaction of PPIX with HRP. Further studies are necessary to elucidate the porphyrin-

induced changes on the enzyme structure involved in enhanced catalytic activity.

ACKNOWLEDGEMENTS

Susmita Sil is a recipient of a Senior Research Fellowship from the University Grants Commission. We thank Prof. U Choudhury for helpful discussions, Prof. B.B. Biswas for making available the spectrophotometer and Prof. C.K Dasgupta for letting us use the spectrofluodmeter.

REFERENCES

[1] : Shopova, M. (1992) in Selected Topics in Photobiology (Jain, V. and Goel, H. eds.), pp. 108-119, Ind. Photobiol. Soc., New Delhi. [2] : Kessel, D. (1984) Photochem. Photobiol. 39, 851- 859. [3] : Wilson, B.D., Mang, T.S. and Stoll, H. (1992) Arch. Dermatol. 128, 1597-1601. [4] : Moan, J. and Berg, K. (1992) Photochem. Photobiol. 55, 931-948. [5] : Daugherty, T.J., Potter, W.R. and Bellnier, D. (1990) in Photodynamic therapy of Neoplastic Disease ( Kessel, D.ed.), pp. 1-19, CRC Press, Boston. [6] : Berns, M.W., Dahtman, A., Johnson, F.M., Burns, R., Sperling, D., Guiltinan, M., Siemens, A., Walter, R., Wright, W., Hammer-Wilson, M. and Wile, A (1982) Cancer Res. 42, 2325-2329. [7]: Bodaness, R.S (1984) Biochem. Biophys. Res Commun 118, 191-197. [8] : van Steveninck, J., Boegheim, J.P.J., Dubbelman, TMA.R. and Van der Zee, J (1987) Biochem J. 242,611-613. [9] : van Steveninck, J., Boegheim, J.P.J., Dubbelman, TM.A.R. and Van der Zee, J. (1988) Biochem. J. 250, 197-201. [10]: Falk, J .E (1964) in Porphyrins & Metalloporphyrins, p.236, Elsevier, Amsterdam. [11]: Takayama, K and Nakano, M (1977) Biochemistry 16, 1921-1926. [12]: Margalit, R., Shaklai, N. and Cohen, S. (1983) Biochem. J. 209, 547-552. [13]: Rotenberg, M. and Margalit, R. (1985) Biochern J. 229, 197-203. [14]: Sil, S. and Chakraborti, A.S. (1996) Ind J. Biochem. Biophys. 33, 285-291. [15]: Kapp, E.A., Daya, S. and Whitley, C.G. (1990) Biochem Biophys. Res. Commun 167, 1383-1392 [16]: Lamola, A.A., Asher, I., Muller-Ebarhard, V. and Poh-Fitzpatric, M (1981) Biochern J. 196, 693-698. [17]: Hirsch, R.E., Lin, M.J., Pulakhandam, UP., Nagel, R.L. and Sandberg, S. (1993) Photochem Photobiol. 57, 885-888. [18]: van Steveninck, J., Dubbelman, T.M.A.R., de Goeij, A F . P . M and Went, L.N (1977) Hemoglobin 1,679-690.

767

Vol. 42, No. 4, 1997 BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL

[19]: Jain, V. (1992) in Selected Topics in Photobiology ( Jain, V. and Goel, H. eds.) pp. 130-147, Ind. Photobiol. Soc., New Delhi. [20]: Martin, A., Swarbrick, J. and Cammarata, A. (1991)in Physical Pharmacy p. 93-113, Varghese Publishing House, Bombay. [21]: Bhattacharyya, J., Bhattacharyya, M., Chakraborti, A.S., Choudhury, U and Poddar, R.K. (1994) Biochem. Pharmacol. 47, 2049-2053. [22]: Martin, A., Swarbrick, J. and Cammarata, A. (1991) in Physical Pharmacy p. 2-26, Varghese Publishing House, Bombay.

768