Journal of Protein Chemistry, VoL 11, No. 5, 1992
The Molybdoenzymes Xanthine Oxidase and Aldehyde Oxidase Contain Fast- and Slow-DTNB Reacting Sulphydryl Groups Francesc Cabr6, L2 Marta Cascante, L3 and Enric I. Canela ~
Received January 31, 1992
The reactivities with an excess of 5-5'-dithiobis (2-nitrobenzoic) acid (DTNB) of sulphydryl residues present in xanthine oxidase and aldehyde oxidase were studied and compared. The results show that two classes of sulphydryl groups with quite different reactivities exist in both enzymes either native or denatured. Some of the available sulphydryl residues thus react instantaneously with the DTNB; whereas the others react very slowly following pseudo-firstorder kinetics. The number of sulphydryl residues of each class and the rate constant of slowly reacting groups are, respectively, 1.7 and 0.8 in native xanthine oxidase and 1.6 ;and t.7 in native aldehyde oxidase. In denatured enzymes, the number of fast- and slow-reacting sulphydryl residues obtained are, respectively, 13.9 and 7.9 in xanthine oxidase and 5.7 and 5.4 in aldehyde oxidase. Analogously, the rate constant for the slowly reacting groups is similar for the two native enzymes, but in denatured aldehyde oxidase it is double that of denatured xanthine oxidase. KEY WORDS: Sulphydryl; DTNB; xanthine oxidase; aldehyde oxidase.
1. I N T R O D U C T I O N
1990; Schaw and Yayatilleke, 1990; Yoshihara and Tatsumi, 1990)o In early studies, the existence of essential thiol groups in xanthine oxidase was observed after inactivating the enzyme by reagents which modify these groups (Green and Massey, 1967). Hille and Massey (1982) detected a disulphide bond at the active site of bovine milk xanthine oxidase, and proposed that this bond can play an important role in the oxidationreduction reaction. In most cases, values between 60 and 62 groups per FAD molecule were obtained in denatured xanthine oxidase. Conversely, only 9.5 available groups were found in the native enzyme (Brumby et al., 1965). The main conclusion is that the availability of the sulphydryl groups depends on experimental conditions. Nevertheless, correlation between reactivity and availability of the sulphydryl groups in xanthine oxidase has not been reported. One of the most recent studies of sulphydryl groups in molybdenum hydroxylases was carried out with chicken liver xanthine dehydrogenase by Canela and
Xanthine oxidase (Xanthine: O2-oxidoreductase, E.C. 1.2.3.2) and aldehyde oxidase (Aldehyde: 02oxidoreductase, E.C. 1.2.3.1) are molybdenum hydroxylases which catalyze oxidation of xanthine to uric acid and aldehydes to acids, respectively. Although the answers to many questions about catalytic aspects of several molybdenum hydroxylases have been found, including kinetic mechanisms (Fonoll et al., 1980; Bruguera et al., 1988) and the structural environment of the active site (Tassayco et al., 1990; Hille et al., 1985; Berg and Holm, 1985; Nishino and Nishino, 1987), their biological function has not been clearly defined to date (Rybczynki et al., 1Department of Biochemistry and Physiology, Faculty of Chemistry, Universityof Barcelona,Marti i Franqubs, I. E-08028 Barcelona, Catalonia, Spain. 2Present address: R&D Department, Menarini Laboratories S.A. Alfonso XII, 587, E-08912 Badalona, Catalonia, Spain. 3To whom all correspondenceshould be addressed. 547
0277-8033/92/t000-0547506.50/0 © 1992 Plenum Publishing Corporation
548 Nin (1985), who found 6.2 and 20.1 available groups per FAD molecule in native and denatured enzyme, respectively. In the present paper, we report a study of sulpbydryl group reactivity of both xanthine oxidase and aldehyde oxidase. We have tested the reactivity of the sulphydryl groups at several enzyme concentrations considering a pseudo-first-order reaction between enzyme and 5,5'-dithiobis (2-nitrobenzoic) acid (DTNB). Our results show that in both enzymes there are two different kinds of detectable groups with quite different reactivities. 2. MATERIALS AND METHODS
2.1. Materials DTNB was obtained from Boehringer Mannheim. All other biochemicals and chemicals were of analytical grade and obtained from Serva, Bio-Rad, Sigma, and Merck. Distilled water, further purified with a Millipore Milli-Q system, was used throughout.
Cabr~ et aL blank was prepared in identical conditions but DTNB was substituted by sodium phosphate buffer. The difference in absorbance was recorded for a minimum of 30 rain at 412 nm. Determination of total (available and hidden) reactive sulphydryl groups was performed after denaturing the enzymes. Thus, 800 pl of enzymic solution of known FAD content (0.5-3 pM) was mixed with 100 pL 20% SDS, and the solution was incubated at 90°C for 15 rain, and rapidly cooled to 20°C. The starting point for incubation time was the beginning of sample heating. The reaction mixture was prepared by addition of 15 pL of 10raM DTNB solution to aliquots of denatured enzyme, previously diluted to 450 p L with 50 mM sodium phosphate buffer, p H 7.8, containing SDS 2% up to the desired concentration of FAD. Blanks contained 50 mM sodium phosphate buffer, p H 7.8, with denaturing reagent instead of DTNB. The reaction was followed under the same conditions described for native enzyme.
2.4. Computational and Statistical Methods 2.2. Purification and Determination of Enzyme Concentration and Activity Purification to homogeneity and determination of concentration as well as activity of both xanthine oxidase and aldehyde oxidase from bovine liver were performed according to Cabr~ and Canela (1987a, b). To avoid the degradation of enzymes, the protease inhibitor PMSF (1.25 raM) was added in the initial steps of the purification. The specific activity of xanthine oxidase and aldehyde oxidase was 3 U/rag and 0.077 U/nag, respectively. Throughout all the experiments, we checked that no polymerization of enzyme molecules had occurred by the formation of disulphide bridges. Enzyme activity was analyzed during the assay with Selwyn's test (Selwyn, 1965), and by determination of enzyme molecular mass by gel filtration in Sephacryl S-300 and electrophoresis in polyacrylamide gels.
2.3. Reactivity of Sulphydryl Groups Reactivity of available thiol groups was studied using a modification of Ellman's method (Ellman, 1957), according to Canela and Nin (1985). The reaction mixture was prepared by addition of 15 p L of 10 mM DNTB to 4 5 0 p L of enzymic solution, containing between 0.5 and 3 p M FAD. An enzyme
All rate equations were analyzed and fitted as well as the parameters calculated and outliers were rejected using a suitable program (Lopez-Cabrera et al., 1988). Discrimination among rival equations was carried out using a method described by Franco et al. (1986). The goodness of the chosen equation was confirmed by the AIC minimum value (Jones et al., 1984), the F-test (Burguillo et al., 1983), and the leastsquares method (Mannervick et al., 1982). All programs were run on a VAX 6310 computer. 3. RESULTS
3.1. Determination of Sulphydryl Groups The classical method for determination of modified sulphydryl groups (Ellman, 1959) cannot discern among different classes of thiols. The reaction can be analyzed, as a first approximation, using the rate equation given by Goldfarb (1966a, b) in the study of the reactivity of protein amino groups with 2,4,6trinitrobenzenesulphonic acid (TNBS) substituting TNBS by DTNB: x=n[FAD](1 - exp(-kt [DTNB]))
(1)
where [DTNB] and [FAD] are molar concentrations, n the number of sulphydryl groups per mole of FAD, x the molar concentration of sulphydryl groups
Fast- and SIow-DTNB Reacting Sulphydryl Groups reacted at time t, and k the rate constant. It should be noted that if 6: is the molar absorptivity of the 2nitro-5-mercaptobenzoate ion, and a, its absorbance at time t, considering that D T N B reacts with free sulphydryl groups liberating 1 mol of 2-nitro-5-mercaptobenzoate anion per mole of thiol, we can substitute x=at/6: in Eq. (1). Then, the number of sulphydryls modified (n) could be determined. However, according to this equation, all the thiol groups would have identical reactivity, that is, the same rate constant k. So, on the basis of the different microenvironments of each sulphydryl residue, it is more convenient to divide sulphydryls into several sets according to their reactivity and availability. In consequence, we can consider m classes of sulphydryls, each set having the same rate constant. Therefore, a more realistic integrated velocity equation is: £/t= 6:
n i [ F A U ] ( 1 - e x p ( - k i t [DTNB]))
(2)
i=l
where n~ is the number of sulphydryl groups per mole of F A D of the ith class, and k; is the rate constant for this ith class. Then, the total number of thiols modified is n. In the particular case that there are two classes of sulphydryl groups, one reacting instantaneously with the D T N B and the other reacting slowly following pseudo-first-order kinetics, Eq. (2) is transformed into: at= [FADI(nl + n2(1 - e x p ( - k a t [DTNB]))) 6:
(3)
549 0.13 ~- ......
g
0.12
i
0.11 z < ~3
g ffl
2 , no further improvement was obtained. According to the method described by Franco et at. (1986), the probability for Eq. (3) was greater than 99.99% for both enzymes and for all the reaction curves. In addition, the goodness of fit of Eq. (3) with respect to the fit of Eq. (2) for n = 1 and n = 2 was supported by three different discrimination tests: the sum of squares (Mannervick, 1982), the Ftest (Burguillo et al., 1983), and AIC (Jones et al.,
3.3. Sulphydryl Groups in Denatured Enzymes As with native enzymes, the goodness of fit of Eq. (3) for both denatured enzymes was demonstrated by means of the tests mentioned above. No dependence o f the number of fast-reacting or slow-reacting sulphydryl groups on protein concentration was detected in either of the denatured enzymes in the range of FAD concentration tested (5.0× l0 -7 to 3.0 × 10 -6 M). A concentration of protein inside the range tested (5.78 × 10 -7 for xanthine oxidase and 1.35 × 10 -6 for aldehyde oxidase) was chosen. As in native enzymes, estimated parameter values were calculated as a weighted average of five reaction curves, which are shown in Table 1.
550
Cabr6 et aL Table I. Available Sulphydryl Groups and Rate Constants for Xanthine Oxidase and Aldehyde Oxidase" nl Native enzyme Denatured enzyme
1.6 4- 0.042 5.7 ± 0.62 nl
Native enzyme Denatured enzyme
Aldehyde oxidase I"/2 1,7 ± 0.10 5.4 ± 0.45
kd
nl + rt2
5.2 ± 0.18 12.2 ± 0.05
3.3 11. !
kd
~'/1-1-H2
3.2±0.54 3.3 ± 0.23
2.5 21.8
Xanthine oxidase n2
1.7±0.19 13.9 ± 0.94
0.82±0.046 7.9 ± 0.82
a Determinations in 50 mM sodium phosphate buffer, pH 7.8, containing EDTA (10 mM), after fitting equation: ~ = [FAD](n, + nz(1 - exp(-kit[DTNB]))) e [SH] is expressed as molar concentrations, and rate constants k~ and k2 in M-'sec-'.
4. DISCUSSION The reaction between sulphydryl groups of xanthine oxidase or aldehyde oxidase and an excess of DTNB apparently shows an exponential dependence on time (Fig. 1). This sort of reaction can be studied according to pseudo-first-order kinetics, applying the hypotheses proposed by Goldfarb (1966a, b). The simplest of these hypotheses implies the a priori assumption that all thiols have the same reactivity. However, not necessaiqly all-the sulphydryl groups react at the same rate, due to different microenvironments. In consequence, the rate equation [Eq. (1)] can be adapted to lead to a mechanistic model consisting of a summation of m equations, each corresponding to sulphydryl groups with similar reactivity [Eq. (2)]. The empirical conclusion based on experimental findings demonstrates that only two classes of thiols can be detected statistically, one reacting almost instantaneously with the DTNB and the other reacting slowly following pseudo-first-order kinetics [Eq. (3)]. The percentage of slow-reacting groups is, therefore, slightly different in the two enzymes (33% for xanthine oxidase and 52% for aldehyde oxidase). Studies on the sulphydryl groups of molybdoenzymes indicates that there are between 3 and 19 groups per hem±molecule in bovine milk xanthine oxidase, 9.5 in porcine liver enz2~ane (Brumby et al., 1965), and 6.7 in chicken liver xanthine dehydrogenase (Canela and Nin, 1985). Our results are similar to these last values. Whereas in native enzymes the total number of SH groups is similar, the number of thiols detected in denatured xanthine oxidase are almost double that for aldehyde oxidase. In addition, ku values increase
when the enzymes are denatured. So, kd for native aldehyde oxidase is approximately 1.6 times greater than the rate constant for native xanthine oxidase, whereas in denatured enzymes it is 3.7 times greater. Available sulphydryl groups after denaturing xanthine oxidase with SDS (21.8/FADmol) were similar to those detected in xanthine dehydrogenase from chicken liver (27.1/FAD mol) (Canela and Nin, 1985), but lower than milk enzyme (62/FAD mol) (Bray and Watts, 1966) a n d h o g liver enzymes (61 and 55/FADmol) (Bray and Malmstrom, 1964; Brumby et al., 1965). The number of thiols modified in denatured aldehyde oxidase (11.1/FAD mol) was lower than in xanthine oxidase. Although the two native enzymes react in a very similar way, they seem to be different after denaturing. In summary, our results suggest that the timecourse analysis of the modification of sulphydryl groups of aldehyde oxidase and xanthine oxidase fits a general empirical equation expressing that native and denatured xanthine oxidase and aldehyde oxidase contain at least two types of thiols: fast- and slowreacting groups, the former reacting almost instantaneously with DTNB. The number of sulphydryl groups available is similar in native enzymes but quite different in denatured enzymes. REFERENCES Berg, J. M., and Holm, R. H. (1985). J. Am. Chem. Soc. 107, 917-925. Bray, R. C., and Watts, D. C. (1966). Biochem. J. 98, 142-148. Bray, R. C., and Malmstrom, B. G. (1964). Biochem. J. 93, 633-634. Bruguera, P., L6pez-Cabrera, A., and Canela, E. I. (1988). Biochem. J. 249, 171-178.
Fast- and Slow-DTNB Reacting Sulphydryl Groups Brumby, P., Miller, R., and Massey, V. (1965). J. Biol. Chem. 240, 2222-2228. Burguillo, J., Wright, A. J., and Bardsley, W. G. (1983). Biochem. J. 211, 23-34. Canela, E. I., and Nin, C. M. (1985). J. Prot. Chem. 4, 305-317. Cabr6, F., and Canela, E. I. (1987a). Biochem. Soc. Trans. 15, 3tl-312. Cabr6, F., and Canela, E. I. (1987b). Biochem. Sac. Trans. 15, 882-883. Ellman, G. L. (1959). Arch. Biochem. Biophys. 82, 70-77. Fonoll, C., Canela, E. I., and Bozal, J. (1980). J. Mol. CataL 8, 401409. Franco, R., Gavald~t, M. T., and Canela, E. I. (1986). Biochem. J. 238, 855-862. Goldfarb, A. R. (1966a). Biochemistry 5, 2570-2574. Goldfarb, A. R. (1966b). Biochemistry 5, 2574-2578. Green, R. C., and O'Brien, P. J. (1967). Biochem. J. 105, 585-589.
551 Hille, R., Hagen, W. R., and Dunham, W. R. (1985). J. Biol. Chem. 260, 10,569-10,575 Hille, R., and Massey, V. (1982). J. BioL Chem. 257, 8898-8901. Jones, R. H., Reeve, E. B., and Swanson, G. D. (1984). Comput. Biomed. Res. 17, 27%288. Lopez-Cabrera, A., Cabr~, F., Franco, R., and Canela, E. I. (1988). Int. J. Biomed. Comput. 23, 9-20. Mannervik, B. (1982). In Methods ofEnzymology (Purich, D. L., ed.), Vol. 87, Academic Press, New York, pp. 370--391. Nishino, T., and Nishino, T. (1987). Biochemistry 26, 3068-3072. Rybczynski, R., Vogt, R. G., and Lerner, M. R. (1990). J. Biol. Chem. 265, 19,712-19,715. Selwyn, M. J. (1965). Bioch#n. Biophys. Aeta 195, 193-I95. Shaw, S., and Jayatflleke, E. (1990). Bioehem. J. 268, 579-583. Tasayeo, M. L., and Prestwich, G. D. (1990). J. Biol. Chem. 265, 3094-3101. Yoshihara, S., and Tatsumi, K. (1990). Drug Metab. Dispos. 18, 676-881.
The Molybdoenzymes Xanthine Oxidase and Aldehyde Oxidase Contain Fast- and Slow-DTNB Reacting Sulphydryl Groups Francesc Cabr6, L2 Marta Cascante, L3 and Enric I. Canela ~
Received January 31, 1992
The reactivities with an excess of 5-5'-dithiobis (2-nitrobenzoic) acid (DTNB) of sulphydryl residues present in xanthine oxidase and aldehyde oxidase were studied and compared. The results show that two classes of sulphydryl groups with quite different reactivities exist in both enzymes either native or denatured. Some of the available sulphydryl residues thus react instantaneously with the DTNB; whereas the others react very slowly following pseudo-firstorder kinetics. The number of sulphydryl residues of each class and the rate constant of slowly reacting groups are, respectively, 1.7 and 0.8 in native xanthine oxidase and 1.6 ;and t.7 in native aldehyde oxidase. In denatured enzymes, the number of fast- and slow-reacting sulphydryl residues obtained are, respectively, 13.9 and 7.9 in xanthine oxidase and 5.7 and 5.4 in aldehyde oxidase. Analogously, the rate constant for the slowly reacting groups is similar for the two native enzymes, but in denatured aldehyde oxidase it is double that of denatured xanthine oxidase. KEY WORDS: Sulphydryl; DTNB; xanthine oxidase; aldehyde oxidase.
1. I N T R O D U C T I O N
1990; Schaw and Yayatilleke, 1990; Yoshihara and Tatsumi, 1990)o In early studies, the existence of essential thiol groups in xanthine oxidase was observed after inactivating the enzyme by reagents which modify these groups (Green and Massey, 1967). Hille and Massey (1982) detected a disulphide bond at the active site of bovine milk xanthine oxidase, and proposed that this bond can play an important role in the oxidationreduction reaction. In most cases, values between 60 and 62 groups per FAD molecule were obtained in denatured xanthine oxidase. Conversely, only 9.5 available groups were found in the native enzyme (Brumby et al., 1965). The main conclusion is that the availability of the sulphydryl groups depends on experimental conditions. Nevertheless, correlation between reactivity and availability of the sulphydryl groups in xanthine oxidase has not been reported. One of the most recent studies of sulphydryl groups in molybdenum hydroxylases was carried out with chicken liver xanthine dehydrogenase by Canela and
Xanthine oxidase (Xanthine: O2-oxidoreductase, E.C. 1.2.3.2) and aldehyde oxidase (Aldehyde: 02oxidoreductase, E.C. 1.2.3.1) are molybdenum hydroxylases which catalyze oxidation of xanthine to uric acid and aldehydes to acids, respectively. Although the answers to many questions about catalytic aspects of several molybdenum hydroxylases have been found, including kinetic mechanisms (Fonoll et al., 1980; Bruguera et al., 1988) and the structural environment of the active site (Tassayco et al., 1990; Hille et al., 1985; Berg and Holm, 1985; Nishino and Nishino, 1987), their biological function has not been clearly defined to date (Rybczynki et al., 1Department of Biochemistry and Physiology, Faculty of Chemistry, Universityof Barcelona,Marti i Franqubs, I. E-08028 Barcelona, Catalonia, Spain. 2Present address: R&D Department, Menarini Laboratories S.A. Alfonso XII, 587, E-08912 Badalona, Catalonia, Spain. 3To whom all correspondenceshould be addressed. 547
0277-8033/92/t000-0547506.50/0 © 1992 Plenum Publishing Corporation
548 Nin (1985), who found 6.2 and 20.1 available groups per FAD molecule in native and denatured enzyme, respectively. In the present paper, we report a study of sulpbydryl group reactivity of both xanthine oxidase and aldehyde oxidase. We have tested the reactivity of the sulphydryl groups at several enzyme concentrations considering a pseudo-first-order reaction between enzyme and 5,5'-dithiobis (2-nitrobenzoic) acid (DTNB). Our results show that in both enzymes there are two different kinds of detectable groups with quite different reactivities. 2. MATERIALS AND METHODS
2.1. Materials DTNB was obtained from Boehringer Mannheim. All other biochemicals and chemicals were of analytical grade and obtained from Serva, Bio-Rad, Sigma, and Merck. Distilled water, further purified with a Millipore Milli-Q system, was used throughout.
Cabr~ et aL blank was prepared in identical conditions but DTNB was substituted by sodium phosphate buffer. The difference in absorbance was recorded for a minimum of 30 rain at 412 nm. Determination of total (available and hidden) reactive sulphydryl groups was performed after denaturing the enzymes. Thus, 800 pl of enzymic solution of known FAD content (0.5-3 pM) was mixed with 100 pL 20% SDS, and the solution was incubated at 90°C for 15 rain, and rapidly cooled to 20°C. The starting point for incubation time was the beginning of sample heating. The reaction mixture was prepared by addition of 15 pL of 10raM DTNB solution to aliquots of denatured enzyme, previously diluted to 450 p L with 50 mM sodium phosphate buffer, p H 7.8, containing SDS 2% up to the desired concentration of FAD. Blanks contained 50 mM sodium phosphate buffer, p H 7.8, with denaturing reagent instead of DTNB. The reaction was followed under the same conditions described for native enzyme.
2.4. Computational and Statistical Methods 2.2. Purification and Determination of Enzyme Concentration and Activity Purification to homogeneity and determination of concentration as well as activity of both xanthine oxidase and aldehyde oxidase from bovine liver were performed according to Cabr~ and Canela (1987a, b). To avoid the degradation of enzymes, the protease inhibitor PMSF (1.25 raM) was added in the initial steps of the purification. The specific activity of xanthine oxidase and aldehyde oxidase was 3 U/rag and 0.077 U/nag, respectively. Throughout all the experiments, we checked that no polymerization of enzyme molecules had occurred by the formation of disulphide bridges. Enzyme activity was analyzed during the assay with Selwyn's test (Selwyn, 1965), and by determination of enzyme molecular mass by gel filtration in Sephacryl S-300 and electrophoresis in polyacrylamide gels.
2.3. Reactivity of Sulphydryl Groups Reactivity of available thiol groups was studied using a modification of Ellman's method (Ellman, 1957), according to Canela and Nin (1985). The reaction mixture was prepared by addition of 15 p L of 10 mM DNTB to 4 5 0 p L of enzymic solution, containing between 0.5 and 3 p M FAD. An enzyme
All rate equations were analyzed and fitted as well as the parameters calculated and outliers were rejected using a suitable program (Lopez-Cabrera et al., 1988). Discrimination among rival equations was carried out using a method described by Franco et al. (1986). The goodness of the chosen equation was confirmed by the AIC minimum value (Jones et al., 1984), the F-test (Burguillo et al., 1983), and the leastsquares method (Mannervick et al., 1982). All programs were run on a VAX 6310 computer. 3. RESULTS
3.1. Determination of Sulphydryl Groups The classical method for determination of modified sulphydryl groups (Ellman, 1959) cannot discern among different classes of thiols. The reaction can be analyzed, as a first approximation, using the rate equation given by Goldfarb (1966a, b) in the study of the reactivity of protein amino groups with 2,4,6trinitrobenzenesulphonic acid (TNBS) substituting TNBS by DTNB: x=n[FAD](1 - exp(-kt [DTNB]))
(1)
where [DTNB] and [FAD] are molar concentrations, n the number of sulphydryl groups per mole of FAD, x the molar concentration of sulphydryl groups
Fast- and SIow-DTNB Reacting Sulphydryl Groups reacted at time t, and k the rate constant. It should be noted that if 6: is the molar absorptivity of the 2nitro-5-mercaptobenzoate ion, and a, its absorbance at time t, considering that D T N B reacts with free sulphydryl groups liberating 1 mol of 2-nitro-5-mercaptobenzoate anion per mole of thiol, we can substitute x=at/6: in Eq. (1). Then, the number of sulphydryls modified (n) could be determined. However, according to this equation, all the thiol groups would have identical reactivity, that is, the same rate constant k. So, on the basis of the different microenvironments of each sulphydryl residue, it is more convenient to divide sulphydryls into several sets according to their reactivity and availability. In consequence, we can consider m classes of sulphydryls, each set having the same rate constant. Therefore, a more realistic integrated velocity equation is: £/t= 6:
n i [ F A U ] ( 1 - e x p ( - k i t [DTNB]))
(2)
i=l
where n~ is the number of sulphydryl groups per mole of F A D of the ith class, and k; is the rate constant for this ith class. Then, the total number of thiols modified is n. In the particular case that there are two classes of sulphydryl groups, one reacting instantaneously with the D T N B and the other reacting slowly following pseudo-first-order kinetics, Eq. (2) is transformed into: at= [FADI(nl + n2(1 - e x p ( - k a t [DTNB]))) 6:
(3)
549 0.13 ~- ......
g
0.12
i
0.11 z < ~3
g ffl
2 , no further improvement was obtained. According to the method described by Franco et at. (1986), the probability for Eq. (3) was greater than 99.99% for both enzymes and for all the reaction curves. In addition, the goodness of fit of Eq. (3) with respect to the fit of Eq. (2) for n = 1 and n = 2 was supported by three different discrimination tests: the sum of squares (Mannervick, 1982), the Ftest (Burguillo et al., 1983), and AIC (Jones et al.,
3.3. Sulphydryl Groups in Denatured Enzymes As with native enzymes, the goodness of fit of Eq. (3) for both denatured enzymes was demonstrated by means of the tests mentioned above. No dependence o f the number of fast-reacting or slow-reacting sulphydryl groups on protein concentration was detected in either of the denatured enzymes in the range of FAD concentration tested (5.0× l0 -7 to 3.0 × 10 -6 M). A concentration of protein inside the range tested (5.78 × 10 -7 for xanthine oxidase and 1.35 × 10 -6 for aldehyde oxidase) was chosen. As in native enzymes, estimated parameter values were calculated as a weighted average of five reaction curves, which are shown in Table 1.
550
Cabr6 et aL Table I. Available Sulphydryl Groups and Rate Constants for Xanthine Oxidase and Aldehyde Oxidase" nl Native enzyme Denatured enzyme
1.6 4- 0.042 5.7 ± 0.62 nl
Native enzyme Denatured enzyme
Aldehyde oxidase I"/2 1,7 ± 0.10 5.4 ± 0.45
kd
nl + rt2
5.2 ± 0.18 12.2 ± 0.05
3.3 11. !
kd
~'/1-1-H2
3.2±0.54 3.3 ± 0.23
2.5 21.8
Xanthine oxidase n2
1.7±0.19 13.9 ± 0.94
0.82±0.046 7.9 ± 0.82
a Determinations in 50 mM sodium phosphate buffer, pH 7.8, containing EDTA (10 mM), after fitting equation: ~ = [FAD](n, + nz(1 - exp(-kit[DTNB]))) e [SH] is expressed as molar concentrations, and rate constants k~ and k2 in M-'sec-'.
4. DISCUSSION The reaction between sulphydryl groups of xanthine oxidase or aldehyde oxidase and an excess of DTNB apparently shows an exponential dependence on time (Fig. 1). This sort of reaction can be studied according to pseudo-first-order kinetics, applying the hypotheses proposed by Goldfarb (1966a, b). The simplest of these hypotheses implies the a priori assumption that all thiols have the same reactivity. However, not necessaiqly all-the sulphydryl groups react at the same rate, due to different microenvironments. In consequence, the rate equation [Eq. (1)] can be adapted to lead to a mechanistic model consisting of a summation of m equations, each corresponding to sulphydryl groups with similar reactivity [Eq. (2)]. The empirical conclusion based on experimental findings demonstrates that only two classes of thiols can be detected statistically, one reacting almost instantaneously with the DTNB and the other reacting slowly following pseudo-first-order kinetics [Eq. (3)]. The percentage of slow-reacting groups is, therefore, slightly different in the two enzymes (33% for xanthine oxidase and 52% for aldehyde oxidase). Studies on the sulphydryl groups of molybdoenzymes indicates that there are between 3 and 19 groups per hem±molecule in bovine milk xanthine oxidase, 9.5 in porcine liver enz2~ane (Brumby et al., 1965), and 6.7 in chicken liver xanthine dehydrogenase (Canela and Nin, 1985). Our results are similar to these last values. Whereas in native enzymes the total number of SH groups is similar, the number of thiols detected in denatured xanthine oxidase are almost double that for aldehyde oxidase. In addition, ku values increase
when the enzymes are denatured. So, kd for native aldehyde oxidase is approximately 1.6 times greater than the rate constant for native xanthine oxidase, whereas in denatured enzymes it is 3.7 times greater. Available sulphydryl groups after denaturing xanthine oxidase with SDS (21.8/FADmol) were similar to those detected in xanthine dehydrogenase from chicken liver (27.1/FAD mol) (Canela and Nin, 1985), but lower than milk enzyme (62/FAD mol) (Bray and Watts, 1966) a n d h o g liver enzymes (61 and 55/FADmol) (Bray and Malmstrom, 1964; Brumby et al., 1965). The number of thiols modified in denatured aldehyde oxidase (11.1/FAD mol) was lower than in xanthine oxidase. Although the two native enzymes react in a very similar way, they seem to be different after denaturing. In summary, our results suggest that the timecourse analysis of the modification of sulphydryl groups of aldehyde oxidase and xanthine oxidase fits a general empirical equation expressing that native and denatured xanthine oxidase and aldehyde oxidase contain at least two types of thiols: fast- and slowreacting groups, the former reacting almost instantaneously with DTNB. The number of sulphydryl groups available is similar in native enzymes but quite different in denatured enzymes. REFERENCES Berg, J. M., and Holm, R. H. (1985). J. Am. Chem. Soc. 107, 917-925. Bray, R. C., and Watts, D. C. (1966). Biochem. J. 98, 142-148. Bray, R. C., and Malmstrom, B. G. (1964). Biochem. J. 93, 633-634. Bruguera, P., L6pez-Cabrera, A., and Canela, E. I. (1988). Biochem. J. 249, 171-178.
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