Journal of Protein Chemistry, Vol. 19, No. 2, 2000

Kinetics of c-Phycocyanin Reaction with Hypochlorite Cheyla Romay,1 Ricardo Gonzalez,1 Marta Pizarro,2 and Eduardo Lissi2,3 Received February 8, 2000

Hydrochlorous acid bleaches c-phycocyanin visible absorbance with a second-order rate constant (pH 7.4) of 1.3 ´ 103 M-1 s-1. In excess of protein, ca. 0.16 bilin moieties are disrupted by each reacted HOCl molecule. This indicates that the main reaction takes place at the apoprotein level, with a total rate constant (in monomeric units concentration) of 2.5 ´ 104 M-1 s-1. This rate constant is too low to provide protection to other biomolecules under physiological conditions. The reported antiinflammatory properties of phycocyanin are not then related to the removal of HOCl. On the other hand, the rather slow reaction rate with HOCl could be beneficial to its role as antiinflammatory agent since it will allow the protein to maintain its integrity at the inflammation locus. KEY WORDS: c-Phycocyanin; hypopchlorite; bilin modification; reaction rate.

1. INTRODUCTION

acid. Several workers have shown that Pc is an efficient singlet oxygen physical quencher, with the bilin group modification only a minor reaction pathway (Tapia et al., 1999). Hypochlorous acid (HOCl) and/or its deprotonated anion (OCl-) play an important role in the immune defense system against microorganisms and also in inflammation. The antiinflammatory action of Pc could then be mediated at least in part by its capacity to remove HOCl. However, this possibility cannot be assessed since there are no data regarding the kinetics and mechanism of Pc interaction with HOCl. To know the interaction rate of the protein with this damaging metabolite it is important to establish its potential capacity to protect other valuable species and/or its ability to remain at the inflammation site (Arouma and Halliwell, 1987) exposed to rather high concentrations of the oxidant. Thermodynamic calculations predict that HOCl is both a one- and two-electron oxidant (Koppenol, 1994), and it has been shown that it readily reacts with thiol and thioether groups (Haenen and Bast, 1991), amines (Marquez and Dunford, 1994), amino acids (Pereira et al., 1973), ascorbate (Folkes et al., 1995), nucleotides (Bernofsky, 1991), and unsaturated hydrocarbons (Winterbourn et al., 1992). Pc can react

c-Phycocyanin (Pc)4 is a protein found in blue-green algae (MacColl and Guard-Friar, 1987). Phycocyanin monomers are themselves made up of two distinguishable protein subunits designated a and b. Each monomer contains three bilin chromophores, open-chain tetrapyrrols without metal complexes (Duerring et al., 1991). The bilin groups are covalently bound to the protein via cysteinyl thioether linkages. The chemical structure of the bilin chromophores is very close to that of bilirrubin, a molecule with well-known antioxidant capacity. Pc has strong antiinflammatory properties in several animal models (Romay et al., 1998; Gonzalez et al., 1999; Remirez et al., 1999; Rimbau et al., 1999; Vadiraja et al., 1998). In most inflammatory processes, associated to PMNL activation and recruiting there is a massive production of ROS, in particular singlet oxygen (from hypochlorite and hydrogen peroxide) and hypochlorous

1

Pharmacology Department, National Center for Scientific Research, CNIC, Havana, Cuba. 2 Chemistry Department, Faculty of Chemistry and Biology, University of Santiago, Santiago, Chile. 3 To whom correspondence should be addressed, at Departmanento de Química, Facultad de Química y Biología, Universidad de Santiago de Chile, Santiago, Chile; e-mail:[email protected]

4

Abbreviations: ABTS, 2,2¢-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt; DTPA, diethylenetriamine pentaacetic acid; HOCl, hypochlorous acid; Pc, c-phycocyanin; PMNL; polymorphonuclear leukocytes; OCl-; hypochlorite anion.

151 0277-8033/00/0200-0151$18.00/0 © 2000 Plenum Publishing Corporation

152

Romay et al.

then at the prosthetic group and apoprotein levels. HOCl has been shown to inactivate a variety of enzymes (Arouma and Halliwell, 1987) with rates that are extremely dependent upon the primary protein structure. In the present work, we have measured the rate of the reaction between Pc and HOCl and evaluated the stoichiometry of the process in terms of the number of bilin groups destroyed per HOCl molecule reacted.

2. MATERIALS AND METHODS HOCl was prepared from commercial Chlorox (5.25% NaOCl). Its concentration was estimated from the solution absorbance at high pH (e = 350 M-1 cm-1, pH 12). Phycocyanin was obtained from Arthospira maxima and purified by the method of Neufeld and Riggs (1969). Catalase from bovine liver (48700 U/mg, less than 0.01% thymol) and DTPA were Sigma products. Measurements were carried out in phosphate buffer 10 mM, pH 7.4. Absorbances were determined in a diode arrange spectrometer (Hewlett Packard 8453) employing a thermostated cubette with magnetic stirring. Disruption of the bilin groups was determined by the change in absorbance at 620 nm. Modification of catalase was evaluated by the decrease in its activity and/or by the decrease in absorbance at 430 nm. Catalase activity was estimated by the rate of hydrogen peroxide consumption. This was evaluated by the rate of absorbance decrease at 250 nm.

Fig. 1. Change in absorption spectra of a solution of c-phycocyanin incubated in the presence of 60 mM HOCl in phosphate buffer, 10 mM, pH 7.4.

3. EXPERIMENTAL RESULTS Addition of HOCl to a Pc solution produces a monotonic decrease in the visible band absorbance (see Fig. 1). This decrease takes place without significant changes in the band shape, thus allowing an estimation of the rate and extent of bilin group destruction. A typical plot of the absorbance at 620 nm versus reaction time is shown in Fig. 2. Both the rate of the process and the remaining absorbance at long reaction times depend, at a given Pc concentration, on the HOCl concentration. At high HOCl/protein ratios, the bleaching of the absorbance is total, indicating that all the bilin groups of Pc are completely disrupted. The plateau observed at long reaction times can then be ascribed to total consumption of the added HOCl. This was further evidenced by addition of extra hypochlorous acid after reaching the plateau, in experiments with low HOCl/protein ratios. A second burst of bilin group consumption takes place immediately after the new HOCl addition.

Fig. 2. Change with time of the absorbance of a c-phycocyanin solution measured at 620 nm following the addition of 60 mM HOCl. Conditions as in Fig. 1.

From the initial slopes of the absorbance decrease it is possible to obtain the rate of the bleaching process. A plot of the initial value of dA/dt against the initial absorbance is shown Fig. 3. These data were obtained at constant HOCl concentration, allowing the evaluation of the order in bilin groups from the slope of the plot. The value obtained was 1.05. Figure 4 shows reaction rate values, obtained at fixed Pc concentrations, plotted as a function of the HOCl concentration. The slope of this plot (0.95) indicates that, over all the concentration range employed, the initial rate law can be expressed as

Phycocyanin and Hypochlorite

Fig. 3. Dependence of the rate of fluorescence bleaching as a function of the initial absorbance of the sample. The rate, expresed as dA/dt, was obtained from the initial slope of an absorbance versus time plot. HOCl concentration was 64 mM in all the experiments. The best straight line shown has a slope of 1.05.

Fig. 4. Dependence of the pseudo first-order rate constant, defined by (dA/dt)/A, as a function of the initial HOCl concentration. The best straight line shown has a slope of 0.95.

Rate of bilin disruption = k(bilin) (HOCl) with k = 1.3 ´ 103 M-1 s-1 at 25°C and pH = 7.4. This is compatible with a simple bimolecular two-electron process for the disruption of the bilin groups by the HOCl/OCl- system. We have changed the pH in the range 6.8–8.0, thus changing the relationship between the protonated and deprotonated hydrochlorous acid (pKa = 7.6). The course of the reaction was similar, and no clear trend in the rate of bilin group disruption was observed. Given the possible change in protonation status of the protein over this pH range, the interpretation of these results is not straight-

153 forward and does not allow us to reach a conclusion regarding whether the active species at physiological pHs is the protonated and/or deprotonated hypo-chorous acid. The spectra given in Fig. 1 show small increases in absorbance at 470, 320, and 250 nm, with clear isosbestic points at 380 and 410 nm. This indicates that the contribution of secondary reactions is minimal, and that most of the products arise from initial protein–HOCl interactions. Simple mass balance considerations then allow us to determine the number of bilin groups destroyed per HOCl reacted. Over all the conditions employed it is obtained that, in excess of Pc, 0.16 ± 0.04 bilin groups are bleached per HOCl molecule introduced into the system. This value implies that bilin groups are not the main targets of the HOCl attack, and that most of the added acid is consumed in processes that do no modify the visible absorption of the bilin moieties. From the rate of bilin group modification and the fraction of reaction at these groups it is concluded that the total rate of Pc reaction with HOCl takes place with a rate constant kPc of (1.3 ´ 3/0.16) ´ 103 M-1 s-1 when the rate is expressed in concentration of Pc monomeric units. Protection of catalase activity has been employed to test the reactivity of different compounds toward HOCl (Arouma, 1997). Several experiments were carried out adding 250 mM HOCl to solutions containing Pc (0.5 mg/ml, 38 mM) and/or catalase (0.3 mg/ml, 5 mM). Modification of catalase can be followed either by the disruption of the heme group (measured by the decrease in absorbance at 430 nm) and/or by the loss of enzymatic activity. At the conditions employed, the rate of the process is too fast to be measured, but the stoichiometry of the process indicates that 0.012 ± 0.003 heme groups are disrupted per HOCl molecule added. Similar values were obtained by measuring the decrease in heme group absorbance or the enzymatic activity loss. This indicates that, relative to Pc, a considerably larger proportion of the HOCl is reacting with the proteic part of the molecule without modifying the integrity of the catalytic site. Experiments carried out employing both proteins indicate mutual protection, as expected from competitive reactions with the added HOCl. Although the error involved in these experiments is rather high, the data indicate that Pc reduces the loss of catalase heme groups by ca. 25%, while catalase reduces bilin group disruption by ca. 35%. If one considers the higher amount of Pc employed in these experiments, it can be concluded that HOCl reacts faster with catalase, and that Pc, at the 0.5 mg/ml level, is not able to completely protect catalase from its reaction with hypochlorous acid.

154 4. DISCUSSION The results obtained in the present work show that the bilin group is not the main target of Pc reaction with HOCl. In this regard, the behavior of Pc is similar to that reported for its reaction with singlet oxygen (Tapia et al., 1999) and contrasts with results obtained employing peroxyl radicals (Lissi et al., 2000) or ABTS radicals (C. Aliaga, unpublished results). In these systems, most of the radicals react with the bilin group and not with the apoprotein. The reaction of the apoprotein with HOCl is relatively slow and, on a milligram basis, slower than that of catalase. This can be related to the lack of free cystein groups in Pc. It has been shown that in proteins, the -SH groups are the most reactive toward HOCl (Arnhold et al., 1991), and that most enzymes are inactivated by oxidation of essential cystein groups (Winterbourn, 1985; Arnhold et al., 1990, 1993). The observed reaction of Pc apoprotein with HOCl can be related to the presence of about nine methionine groups in this molecule (Schirmer et al., 1986). Methionine groups also efficiently react with HOCl (Arnhold et al., 1991), and the inactivation of a1-antiproteinase has been associated with an oxidation of methionine residues. The reaction of the bilin group could also result from an oxidation of the ether linkage that binds the chromophore to the apoprotein (Arnhold et al., 1990). However, the total bleaching of the visible absorbance associated with the reaction at the prosthetic group level would favor an oxidation of the double bond system of the tetrapyrrol system, as found in its reaction with singlet oxygen (Braslavsky et al., 1991). Regarding the possible removal of HOC1 by Pc as part of its function as antiinflammatory agent, we consider that the rate of the process is too slow to significantly reduce the damage associated with HOCl in inflammatory processes. In fact, the reaction rate found in the present work is several orders of magnitude smaller than that of other biomolecules, such as ascorbic acid and glutathione (Folkes et al., 1995). Also, the small degree of protection afforded to catalase by rather high (0.5 mg/ml) concentrations of Pc found in the present work argues against an antiinflammatory action based on the removal of HOCl. On the other hand, a rather slow rate of reaction of Pc toward HOCl and singlet oxygen (Tapia et al., 1999) could be beneficial, since it will allow the protein to maintain its integrity at the inflammation locus.

ACKNOWLEDGMENTS This work was supported by FONDECYT (1970691) and a Catedra Presidential en Ciencias, 1996.

Romay et al. REFERENCES Arnhold, J., Hammerschmidt, S., Wagner, M., Mueller, S., Arnold, K., and Grimm, E. (1990). On the action of hypochlorite on human serum albumin. Biomed. Biochim. Acta 49, 991–997. Arnhold, J., Hammerschmidt, S., and Arnold, K. (1991). Role of functional groups of human plasma and luminol in scavenging of NaOCl and neutrophil-derived hypochlorous acid. Biochim. Biophys. Acta 1097:145–151. Arnhold, J., Mueller, S., Arnold, K., and Sonntag, K. (1993). Mechanisms of inhibition of chemiluminescence in the oxidation of luminol by sodium hypochlorite. J. Biolumin. Chemilumin. 6, 307–313. Aruoma, O. I. (1997). Scavenging of hypochlorous acid by carvedilol and ebselen in vitro. Gen. Pharmacol. 28, 269–272. Aruoma, O. I., and Halliwell, B. (1987). Action of hypochlorous acid on the antioxidant protective enzymes superoxide dismutase, catalase and gluthatione peroxidase. Biochem. J. 248, 973–976. Bernofsky, C. (1991). Nucleotide chloramines and neutrophil-mediated cytotoxicity. FASEB J. 5, 295–300. Braslavsky, S. E., Schneider, D., Heihoff, K., Nonell, S., Aramendia, P. F., and Schaffner, K. (1991). Phytochrome models. 11. Photophysics and photochemistry of phycocyano bilin dimethyl ester. J. Am. Chem. Soc. 113, 7322–7334. Duerring, M., Schmidt, G. B, and Huber R. (1991). Isolation, crystallization, crystal structure analysis and refinement of constitutive c-phycocyanin from chromatically adapting cyanobacterium Fremyella diplosiphon at 1.66 Å resolution. J. Mol. Biol. 217, 577–592. Folkes, L. K., Candeias, L. P., and Wardman, P. (1995). Kinetics and mechanisms of hypochlorous acid reactions. Arch. Biochem. Biophys. 20, 120–126. González, R., Rodríguez, R. S., Romay, Ch., Ancheta, O., González, A., Armesto, J., Remirez, D., and Merino, N. (1999). Antiinflammatory activity of phycocyanin extract in acetic acid induced colitis in rats. Pharmacol. Res. 39, 55–59. Haenen, G. R. M. M., and Bast, A. (1991). Scavenging of hypochlorous acid by lipic acid. Biochem. Pharmacol. 42, 2244–2246. Koppenol, W. H. (1994). Thermodynamic considerations on the formation of reactive species from hypochlorite, superoxide and nitrogen monoxide. Could nitrosyl chloride be produced by neutrophils and macrophages? FEBS Lett. 347, 5–8. Lissi, E. A., Pizarro, M., Aspée, A., and Romay, Ch. (2000). Kinetics of phycocyanin bilin groups destruction by peroxyl radicals. Free Rad. Biol. Med., (in press). MacColl, R., and Guard-Friar, D (1987). Phycobiliproteins, CRC Press, Boca Raton, Florida. Marquez, L. A., and Dunford, H. B. (1994). Chlorination of taurine by myeloperoxidase J. Biol. Chem. 269; 7950–7956. Neufeld, G. J., and Riggs, A. F. (1969). Aggregation properties of c-phycocyanin from Anacystis nidulans. Biochem. Biophys. Acta 181, 234–243. Pereira, W. E., Hoyano, Y., Summons, R. E., Bacon, V. A., and Duffield, A. M. (1973). Chlorination studies. II The reaction of aqueous hypochloruos acid with a-amino acids and dipeptides. Biochim. Biophys. Acta 313, 170–180. Remirez, D., González, A., Merino, N., González, R., Ancheta, O., Romay, Ch., and Rodríguez, S. (1999). Effect of phycocyanin in zymosaninduced arthritis in mice. Drug Dev. Res. 48, 70 –75. Rimbau, V., Camins, A., Romay, Ch., González, R., and Pallas, M. (1999). Protective effects of phycocyanin against kainic acidinduced neuronal damage in rat hippocampus. Neurosci. Lett. 276, 75 –78. Romay, C., Ledón N., and González, R. (1998) Further studies on antiinflammatory activity of phycocyanin in some animal models of inflammation. Inflamm. Res. 47, 334–338. Schirmer, T., Huber, R., Schneider, M., Bode, W., Miller, M., and Hackert, M. L. (1986). Crystal structure analysis and refinement at 2.5 Å of hexameric c-phycocyanin from the cyanobacterium Agmenellum quadruplicatum. The molecular model and its implications for light-harvesting. J. Mol. Biol. 188, 651–676.

Phycocyanin and Hypochlorite Tapia, G., Galetovic, A., Lemp, E., Pino, E., and Lissi, E. A. (1999) Singlet oxygen mediated photobleaching of the prosthetic group in hemoglobins and c-phycocyanin. Photochem. Photobiol. 70, 499–504. Vadiraja, B. B., Gaikwad, N. W., and Madyastha, K. M. (1998). Hepatoprotective effect of phycocyanin: Protection for carbon tetrachloride and R-(+)-pulegone-mediated hepatotoxicity in rats. Biochem. Biophys. Res. Commun. 249, 428–431.

155 Winterbourn, C. C. (1985). Comparative reactivities of various biological compounds with myeloperoxidase-hydrogen peroxide-chloride, and similarities of the oxidant hypochlorite. Biochim. Biophys. Acta 840, 204–210. Winterbourn, C. C., van den Berg, J. J. M., Roitman, E., and Kuypers, F. A. (1992). Chlorohydrin formation from unsaturated fatty acids reacted with hypochlorous acid. Arch. Biochem. Biophys. 296, 547–555.