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Studies on the chemical modification of hemorrhagic toxin I from five pace snake (Agkistrodon acutus) venom
Taxkon, Vol. 23, No . 2, pp . 283-288, 1985 . Rioted in drat Hritaio.
0041-0101/83 ß .00+ .00 O 1983 PerBamoo Press Ltd .
STUDIES ON THE CHEMICAL MODIFICATION OF HEMORRHAGIC TOXIN I FROM FIVE PACE SNAKE (AGKISTRODON ACUTUS) VENOM XUN XU, YAN-HUA ZHU, YU-ZHEN WANG, YANG MA and ZI-XIAN LUD Department of Biology, University of Science and Technology of China, Hefei, Anhui, People's Republic of China (Accepted for publication 25 September 1984) X . Xu, Y . H . ZHU, Y. Z . W~rlc, Y. Mw and Z . X . Lu . Studies on the chemical modification of hemorrhagic toxin I from five pace snake (Agkistrodon ocular) venom . Te~xicon 23, 283 - 288, 1985 . - The chemical modification of hemorrhagic toxin I (AaHI) from Agkistrodon ocular has ban studied . Inactivation was observed upon modification of 3 out of 7 histidine residues with diethyl pyrocarbonate . The His residues are deblocked, accompanied by a return of activity, upon treatment with neutral hydroxylamine . The circular dichroism and fluorescence spectra of diethyl pyrocarbonate inactivated toxin and the native toxin are the same, indicating that modification with diethyl pyrtx:arbonate does not cause any gross change in the structure of the protein . At least one His residue may thus play an essential role in the enzyme activity . Reaction of the toxin with N-bromosuccimide abolished the enzyme activity, with modification of Trp, Tyr and His residues. The loss of Trp did not parallel the inactivation . Hydrogen peroxide, dioxane and 2-hydroxy-Snitrobenzyl-bromide treatment damaged the Trp residues, but did not affect the activity . Therefore, the modified tryptophan side chains are not essential for activity . Modification of 2 .5 - 3 .0 Tyr residues out of 9 with acetylimidazole did not affect the enzyme activity, nor did nitration of the toxin with tetranitromethane . The reactive tyrosines are apparently not essential .
INTRODUCTION WE HAVE reported the purification and some properties of hemorrhagic compounds from A. ocular venom (Xu et al., 1981) . One of them, designated AaHI, is an acidic protein and possesses caseinolytic activity . Its molecular weight is 22,000 and its pI is 4.6. The enzyme activity is optimal at alkaline pH and is inhibited by EDTA . It is known that proteases are present in various snake venoms, especially in those of Crotalidae and Viperidae . Some of the proteases are related to the hemorrhagic and myonecrotic activities (BJARNASON and TU, 1978 ; TU, 1982) . Despite their widespread distribution and importance to biological activities, proteases from snake venoms remain poorly understood with respect to structure and function, especially compared to what is known about such well-studied proteolytic enzymes as serine proteases or metallocarboxypeptidases . In this paper, we report the results of chemical modification of AaHI with diethyl pyrocarbonate (ethoxy formic anhydride (DEP), N-bromosuccimide (NBS), 2-hydroxy-5-nitrobenzyl bromide (HNB-bromide) and tetranitromethane (TNM). MATERIALS AND METHODS Materials. AsHI was purified from crude A . ocular venom by a procedure described previously (Xu et al., 1981) . NBS and N-acetylimidazole were purchased from F7uka, HNB-bromide and DEP from Sigma, and TNM was generously provided by Dr Xu Geng Jun of the Shanghai Institute of Biochemistry . Casein was purchased " Permanent address : Shanghai Institute of Biochemistry, Academic Sinisa, Shanghai . 283
284
XUN XU tt al.
from E. Merck, Germany, and Sephadex G-23 from Pharmacia, Swedrn . All other chemicals used in this study were analytical grade or equivalent . Mod(Ccation with DEP. stock solutions of DEP were prepared by dilution with ice-cold absolute ethanol. AaHI (2 .3 x 10 - ' M) was incubated with the indicated concentration of DEP at 20°C for 30 min. The concentration of N~arbethoxyimidazole formed was determined from the increase in absorbance at 240 nm, assuming an absorbency of 3 .2 x ld M - ' cm - ' (Muss, 1977). The reacüon with DEP was reversed by treatment with 0.4 M NH,OH (pH 7 .0) at S°C for 12 hr . The protein was then separated from the reagrnts by passage through a Sephadex G-25 column . Reaction with N-acetyllmidazole. Atetylation was performed according to the method of Rtoxnnw and V~t,t.ee (1972) . Nitration with TNM. Nitration of AaHI with TNM was carried out at pH 8.0 in 0.05 M Tris buffer (Rtoannn and Vni.>.e~, 1972). TNM was diluted 1 :10 with 95s7o ethanol. Aliquots of TNM in 95aK ethanol were added to AaHI and the reaction was performed at room temperature for 90 min. Mod cation with HNB-bromide. Reaction with HNB-bromide was carried out at room temperature (20°C) . In atypical experimrnt, a total of 30 kl solution of HNB-bromide in dry acetone was added dropwise into 3.0 ml of a 1 mg/ml solution of AaHI in 0.1 M sodium acetate buffer (pH 6.0) with constant stirring. The pH was maintained at 6.0 by the addition of 1 .0 N NaOH . After 30 min the modified protein was separated from excess reagrnt on a Sephadex G-25 (1 .0 x 20 cm) column equilibrated with the same buffer . The number of moles of HNB bound per mole of AaHI was measured spectrophotometrically (BARMAN and KosHt.Arm 1%7 ; absorbency at 410 nm, 18,000 M- ' cm - ') . Oxidation of AaHI by H,O,-dioxane . AaHI (5 x 10 - ' M) in 2.0 ml of 0.5 M NaHCO, buffer (pH 8.4) was oxidized with various concentration of H,O,-dioxane, prepared aaording to the procedure of HwctnMOa~ et al. (1964) at S°C for 2.5 hr . The progress of the oxidation was followed by observing thechange in absorbency at 280 nm, which was calculated to be 3.49 x 10' (HACttiMOx~ et al., 1964). Modjfcation with NBS . This modification was performed according to theprocedure of $PANDS et al. (1966) . In a typical experimrnt the concentration of AaHI was S x 10 - ' M. Both the enzyme and reagentwere dissolved in 0.1 M sodium acetate buffer (pH 6.0). The reagrnt/AaHI molar ratio used varied from 2 to 40. After 30 min reaction at 20°C, an aliquot of the modified AaHI solution was withdrawn for determination of caseinolytic activity and hemorrhagic activity, respectively . The number of tryptophan residues oxidized was determined by amino acid analysis . Circubr dichroism andfluorescence spectra. CD spectra were measured with aJasco J-500 dichropohuimeter (Japan). The light path of the couches used was 0.3-0 .05 cm . All the data are expressed as molar residual ellipticity. The mean residue weight of AaHI was takrn as 110. The fluorescence spectrum of AaHI was measured with a Shimadzu RF Spectrofluorometer (Japan). The wavelength of excitation was 295 nm . The fluorescence intrnsitiea of AaHI were expressed as relative fluorescence intensity. Amino acid analysis . Analysis was performed with an automatic amino acid analyzer (Hitachi model 833 analyzer). Protein hydrolysis were carried out in 5.7 N HCl at 110°C under nitrogrn for 24 hr . Tryptophan was determined after hydrolysis with 4N p.tolurnesulphonic acid (Ltu and CtaAtvo, 1971). Enryme assay and analytical methods. Caseinolytic activity was measured colorimetrically according to the method of MIJaAi'A et al. (1%3). Hemorrhagic activity was determined by the method of Koxoo et al. (1960) . Immunoprxipitation was conducted in 1~ aqueous agar by the Ouchterlony method of double diffusion with antitoxic horse serum. Protein concentration was determined spectrophotometrically, assuming an absorbante of 1 .2 for 0.1% AaHI at 280 nm . RESULTS
Mod cation with DEP
The effects of various concentrations of DEP on enzyme activity are shown in Fig. 1 . The progress of oxidation was followed by measuring the change in absorbency at 240 nm (Fig. 1). The extent of modification of His residues was calculated from the increase in absorbance at 240 nm of the reaction solution (A~=3.2 x 10' M- ' cm - ') (MILES, 1977). The correlation between the degree of AaHI inactivation and the number of His modified by DEP is shown in Fig. 2. The result of amino acid analysis (data not shown) was consistent with that from spectrometric method . The results indicated that when approximately three residues of His per molecule of enzyme reacted with DEP, AaHI lost 85% of its original activity, not only its caseinolytic activity, but also its hemorrhegic and immunochemical activities (data not shown) . Most of the activity lost could be recovered upon treatment of the modified enzyme with NHZOH. Incubation in the absence of NH20H did not cause any restoration of activity .
Chemical Modification of AaHI
28 5
100 0.30
eo
0.20 {+
U O
y tr
40
0 aN
0 .10
0
0 1 10
I 0
I 20
I 30
(mmole)
DEP
I 40
Flo. 1 . REACTION OF DIETHYL PYROCARBONATE (DEP) wITH HEMORRHAOgc roxnv I (AaHI) . AaHI (2 .5 x 10-' M) was incubated with the indicated concentration of DEP at 20°C . The histidine reaction was revenged for 30 min by treatment with 0.4 M NH~OH (pH 7.0) at 5°C for 12 hr . " A increase in absorbance at 240 nm ; Q - _ _ _ _ _ O - " Enzyme activity; A absorbance at 240 nm of modified AaHI after treatment with NH~OH at pH 7.0.
Circular dichroism and fluorescence were also used to investigate the conformational aspects of modified AaHI. The CD spectra of DEP-inactivated AaHI and native AaHI are quite similar in the far u.v. region (Fig. 3). The maximum of the emission spectrum of AaHI was around 350 nm, and after AaHI was inactivated by DEP the emission maximum was still 350 nm, with a 30% diminution of intensity (Fig. 4). This indicated that only minor changes in tertiary structure had occurred . Since modification of Trp by DEP causes complete loss of fluorescence (ROSEN et al., 1970), it was apparent that not more than one Trp could have been modified by DEP. Inhibition of AaHl by NBS The effect of various concentration of NBS on caseinolytic activity is shown in Table 1 . A 30 fold molar excess of NBS over AaHI decreased the proteolytic activity by 80%. A similar result was observed for the hemorrhagic activity (data not shown) . Of the original activity, SO% remained after modification of all three tryptophan residues (Table 1). Although NBS has been shown to modify Trp residues in protein with considerable specificity ($PANDE et al., 1966). Tyrosine, histidine and methionine can also react with loor.\
?~ eo r
*~ so o p0
v
i
0 20 0
I 0
I I
I 2
\I 3
HIS residues modified / mol
FIa . 2. CORRELATION HETWF.EN INACTIVATION OF AaHI AND THE NUMBER OF HIS RESIDUES MODIFIED . AaHI (5 x 10 -' M) in 0.1 M acetate buffer (pH 6.0) was treated with various concentration of DEP at 20°C for 60 min. The number of His residues modified was calculated as described in the text, according to the method of SFANDE and WTTKOF (197n.
I 210
I 220
a
I 230 lnm)
I 240
I 250
AaHI . Native AaHI (0 .1 mg/ml) in 0.01 M acetate buffer (pH 6.0); - - - - - - AaHI plus 30 mM DEP after 30 min at 20°C; . . . . . . . . . . AaHI plus N-bromosuccimide (NBS/AaHI=30/1) after 30 min at 20°C . FIG . 3 . CIRCULAR DICHROISM SPECTRA OF
NBS (SHALTIEL and PATCHORNIK, 1963), especially at pH 6.6. The numbers of these amino acids from AaHI at various stages of oxidation are shown in Table 1 . Tyr and His decreased gradually during the treatment with NBS. Modification of AaHI with HNB-bromide, H~O~-dioxane, acetylimidazole and TNM Upon incubation of AaHI for 30 min with HNB-bromide (60 mM - 240 mM), Trp became modified ; 240 mM reagent resulted in the modification of 3 Trp residues, as determined both spectrophotometrically and by amino acid analysis . No loss of enzyme activity was observed . loo T N C N C N U C
ar " +
0
m
300
350
a
400
450
lnm)
FIG . 4 . FLUORESCENCE EMISSION SPECTRA OF ABHI . Native AaHI (3 x 10 -" M) in 0.01 M acetate buffer (pH 6.0); - - - - - - AaHI plus 30 mM DEP after 30 min at 20°C ; . . . . . . . . AaHI plus NBS (NBS/AaHI=30/1) after 30 min at 20°C . The fluorescence intensities of AaHI are expressed as relative fluorescence intensity. Excitation wavelength 295 nm .
Chemical Modification of AaHI
28 7
TABLE 1 . EFFECT OF OXIDATION OF ABHI (HEMORRFIAGIC TOXIN I) BY NBS (N BROMOS000IMIDE) ON ENZYMATIC ACTIVITY AND ON NUMBERS OF REACTIVE AMINO ACID RESIDUES
Molar excess
Amino acid "
Trpt Tyr Met His enzyme activity (%)*
NBS
0
4
l2
IS
20
30
3.2 9 4 7
2 .3 8 4 7
0.64 6 4 6
0 4 4 5
0 3 3.7 4
0 0 4 3.5
100
100
60
60
28
23
"The composition of other amino acids is not shown in the table for simplicity, as they were the same as those of the native enzyme . Trp was determined after hydrolysis by 4 N p-toluenesulphonic acid for 24 hr . *The actual absolute 100% value is 48 units. (The unit is defined as Ng tyrosine equivalent of TCA soluble product formed per min per mg protein.)
The result of oxidation of AaHI by H20z - dioxane was similar to that of HNB modification . Oxidation of AaHI was carried out with various concentrations of H202 -dioxane at pH 8.4 in 0.5 M NaHCO, buffer for 2.5 hr at 5°C. After incubation with 100 mM and 2fW mM HZOZ -dioxane 1 .13 and 2.6 out of 3 Trp residues were modified, respectively, but no change in enzyme activity was observed . Incubation of AaHI sodium barbital buffer, pH 7.5 for 30 min at 4°C in a pH-stat with a 200-fold molar excess of acetylimidazole resulted in the modification of 3 Tyr residues out of 9 present in AaHI . A S% loss of enzyme activity was observed, therefore modification of three Tyr residues in AaHI does not seem to influence its enzyme activity . The reaction of AaHI with TNM caused no loss of activity . DISCUSSION
Diethyl pyrocarbonate (DEP) is used for modification of the imidazole group. In the presence of 30 mM DEP, the enzyme activity decreased by 80%, accompanied by modification of 2 - 3 His residues out of the 7 present in Aa.HI. The loss of activity can be reversed by removing the carbethoxy group from the modified imidazole. Circular dichroism and fluorescence studies indicated that the modified enzyme retained its native conformation . Therefore, it could be deduced that the loss of the activity might be directly correlated with the modification of the His side chans). The extrapolated data shown in Fig. 2 indicated that'modification of three His residues abolished the activity of AaHI . It seems unlikely, however, that all three His residues could be involved in the active site of a protease . Considering that in a protein molecule some side chains have the same chemical potential to be modified, we can only deduce that at least one His residue is involved in the active site of the AaHI molecule . A gradual loss of activity was observed when AaHI was treated with NBS, which is a specific reagent for the modification of the Trp side chain at pH 4.5. It was found, however, that under the conditions used here, Tyr and His side chains of AaHI were modified simultaneously. In order to correlate the function of tryptophan side chains with the activity of AaHI, more evidence was necessary. The results given demonstrate that AaHI remains fully active upon oxidation of 2.6 Trp residues with HZOZ - dioxane. This
288
XUN XU
et al.
suggests that the side chains of Trp of AaHI are not essential for function . That HNBbromide treatment of AaHI did not damage the activity, supports this conclusion . It seems likely that Tyr side chains are also non-essential for the activity of AaHI, since the modification of Tyr with acetylimidazole did not affect AaHI activity . The finding that the treatment of AaHI with TNM had no effect on its activity supports this suggestion. We therefore conclude that one or two His residues) are involved in the active site of AaHI, while Trp and the reactive Tyr side chains are not essential for its activity . The modification of the essential His side chain may account for the loss of activity observed upon treatment with NBS, however, the active center of AaHI requires further study. Acknowledgment - we
thank Dr Xu GENG Jux for the gift of TNM. REFERENCES
B~tttitnrr, T. E. and Kosat.nxn, D. E. (1%7) A colorimetric procedure for the quantitatvn determination of tryptophan residues in protein. J. biol . Chem . 242, 5771 . B~Nex~sox, J. B. and Tu, A. T. (1978) Hemorrhagic toxins from western diamondback rattlesnake (Crotalus atrox) venom: isolation and characterization of five toxins and the role of zinc in hemorrhagic toxin e. Biochemistry 17, 3395 . HACHIMORI, Y., Hoatxtsxt, L, Kutttttnaw, K. and $HIBATA, K. (1964) Studies of amino acid residues in proteins . V. Different reactivities with H,O, of tryptophan residues in lysozyme, proteins and zymogens . Biochim. biophys. Acta 93, 346. Koxno, H., Koxno, S., IKezAWn, H., MURATA, K. and GHSnxn, A. (1960) Studies on the quantitative methods for determination of hemorrhagic activity of habu snake venom. Jap. J. med. Sci. Biol. 13, 43 . Liu, T. Y. and Cttvvo, Y. H. (1971) Hydrolysis of protein with p-toluenesulfonic acid determination of tryptophan . J. biol. Chem . 246, 2842 . M~1.ES, E. W. (1977) Modification of histidyl residues in proteins by diethylpyrocarbonate In : Methods in Enzymology, Vol. 47, 431 (H~xs, C. H. W. and TIMnsHEeF, S. N., Eds) . New York : Academic Press. MURATA, Y., SATAKE, M. and Suzuxt, T. (1%3) Studies on snake venom . XII. Distribution of proteinase activities among Japanese and Formosan snake venom. J. Biochem. 53, 431 R~ottn~+rt, J. F. and V.~t .LEe, B. C. (1972) 0 .acetyltyrosine . In : Methods in Enzymology, Vol. 25, p. 500 (H~xs, C. H. W. and TtMASHEFF, S. N., Eds) New York : Academic Press. RIORDAN, J. F. and VAL.LEE, B. C. (1972) Nitration with tetranitromethane. In : Methods in Enyymology, Vol. 25, p 515 (H~xs, C . H . W. and TtMast-~r-~, S. N., Eds) New York : Academic Press. Rosew, C. G., GEVALL, T. and AxnExssox, L. O. (1970) Reaction of diethylpyrocarbonate with indole derivatives with special reference to the reaction with tryptophan residues in a protein. Biochim. biophys. Acta 221, 207. SHALTIEL, S. and PALTCHORNIK, A. (1%3) Cleavage of histidyl peptide bonds by N-bromosuccinimide. J. Am . chem. Soc. 85, 2799 . SPANDE, T. F., GREEx, N. M. and WITKOI', B. (1966) The reactivity toward N-bromosuccinimide of tryptophan in enzymes, zymogens and inhibited enzymes. Biochemistry S, 1926. Tu, A. T. (1982) Hemorrhage induced by snake vcnoms . J. Formosan med. Ass. 81, 807. Xu, X., Wntvo, C. Ltu, J. and Lu, Z. (1981) Purification and characterization of hemorrhagic components from Agkistrodon acutus (hundred pace snake) venom Toxicon 19, 633 .
0041-0101/83 ß .00+ .00 O 1983 PerBamoo Press Ltd .
STUDIES ON THE CHEMICAL MODIFICATION OF HEMORRHAGIC TOXIN I FROM FIVE PACE SNAKE (AGKISTRODON ACUTUS) VENOM XUN XU, YAN-HUA ZHU, YU-ZHEN WANG, YANG MA and ZI-XIAN LUD Department of Biology, University of Science and Technology of China, Hefei, Anhui, People's Republic of China (Accepted for publication 25 September 1984) X . Xu, Y . H . ZHU, Y. Z . W~rlc, Y. Mw and Z . X . Lu . Studies on the chemical modification of hemorrhagic toxin I from five pace snake (Agkistrodon ocular) venom . Te~xicon 23, 283 - 288, 1985 . - The chemical modification of hemorrhagic toxin I (AaHI) from Agkistrodon ocular has ban studied . Inactivation was observed upon modification of 3 out of 7 histidine residues with diethyl pyrocarbonate . The His residues are deblocked, accompanied by a return of activity, upon treatment with neutral hydroxylamine . The circular dichroism and fluorescence spectra of diethyl pyrocarbonate inactivated toxin and the native toxin are the same, indicating that modification with diethyl pyrtx:arbonate does not cause any gross change in the structure of the protein . At least one His residue may thus play an essential role in the enzyme activity . Reaction of the toxin with N-bromosuccimide abolished the enzyme activity, with modification of Trp, Tyr and His residues. The loss of Trp did not parallel the inactivation . Hydrogen peroxide, dioxane and 2-hydroxy-Snitrobenzyl-bromide treatment damaged the Trp residues, but did not affect the activity . Therefore, the modified tryptophan side chains are not essential for activity . Modification of 2 .5 - 3 .0 Tyr residues out of 9 with acetylimidazole did not affect the enzyme activity, nor did nitration of the toxin with tetranitromethane . The reactive tyrosines are apparently not essential .
INTRODUCTION WE HAVE reported the purification and some properties of hemorrhagic compounds from A. ocular venom (Xu et al., 1981) . One of them, designated AaHI, is an acidic protein and possesses caseinolytic activity . Its molecular weight is 22,000 and its pI is 4.6. The enzyme activity is optimal at alkaline pH and is inhibited by EDTA . It is known that proteases are present in various snake venoms, especially in those of Crotalidae and Viperidae . Some of the proteases are related to the hemorrhagic and myonecrotic activities (BJARNASON and TU, 1978 ; TU, 1982) . Despite their widespread distribution and importance to biological activities, proteases from snake venoms remain poorly understood with respect to structure and function, especially compared to what is known about such well-studied proteolytic enzymes as serine proteases or metallocarboxypeptidases . In this paper, we report the results of chemical modification of AaHI with diethyl pyrocarbonate (ethoxy formic anhydride (DEP), N-bromosuccimide (NBS), 2-hydroxy-5-nitrobenzyl bromide (HNB-bromide) and tetranitromethane (TNM). MATERIALS AND METHODS Materials. AsHI was purified from crude A . ocular venom by a procedure described previously (Xu et al., 1981) . NBS and N-acetylimidazole were purchased from F7uka, HNB-bromide and DEP from Sigma, and TNM was generously provided by Dr Xu Geng Jun of the Shanghai Institute of Biochemistry . Casein was purchased " Permanent address : Shanghai Institute of Biochemistry, Academic Sinisa, Shanghai . 283
284
XUN XU tt al.
from E. Merck, Germany, and Sephadex G-23 from Pharmacia, Swedrn . All other chemicals used in this study were analytical grade or equivalent . Mod(Ccation with DEP. stock solutions of DEP were prepared by dilution with ice-cold absolute ethanol. AaHI (2 .3 x 10 - ' M) was incubated with the indicated concentration of DEP at 20°C for 30 min. The concentration of N~arbethoxyimidazole formed was determined from the increase in absorbance at 240 nm, assuming an absorbency of 3 .2 x ld M - ' cm - ' (Muss, 1977). The reacüon with DEP was reversed by treatment with 0.4 M NH,OH (pH 7 .0) at S°C for 12 hr . The protein was then separated from the reagrnts by passage through a Sephadex G-25 column . Reaction with N-acetyllmidazole. Atetylation was performed according to the method of Rtoxnnw and V~t,t.ee (1972) . Nitration with TNM. Nitration of AaHI with TNM was carried out at pH 8.0 in 0.05 M Tris buffer (Rtoannn and Vni.>.e~, 1972). TNM was diluted 1 :10 with 95s7o ethanol. Aliquots of TNM in 95aK ethanol were added to AaHI and the reaction was performed at room temperature for 90 min. Mod cation with HNB-bromide. Reaction with HNB-bromide was carried out at room temperature (20°C) . In atypical experimrnt, a total of 30 kl solution of HNB-bromide in dry acetone was added dropwise into 3.0 ml of a 1 mg/ml solution of AaHI in 0.1 M sodium acetate buffer (pH 6.0) with constant stirring. The pH was maintained at 6.0 by the addition of 1 .0 N NaOH . After 30 min the modified protein was separated from excess reagrnt on a Sephadex G-25 (1 .0 x 20 cm) column equilibrated with the same buffer . The number of moles of HNB bound per mole of AaHI was measured spectrophotometrically (BARMAN and KosHt.Arm 1%7 ; absorbency at 410 nm, 18,000 M- ' cm - ') . Oxidation of AaHI by H,O,-dioxane . AaHI (5 x 10 - ' M) in 2.0 ml of 0.5 M NaHCO, buffer (pH 8.4) was oxidized with various concentration of H,O,-dioxane, prepared aaording to the procedure of HwctnMOa~ et al. (1964) at S°C for 2.5 hr . The progress of the oxidation was followed by observing thechange in absorbency at 280 nm, which was calculated to be 3.49 x 10' (HACttiMOx~ et al., 1964). Modjfcation with NBS . This modification was performed according to theprocedure of $PANDS et al. (1966) . In a typical experimrnt the concentration of AaHI was S x 10 - ' M. Both the enzyme and reagentwere dissolved in 0.1 M sodium acetate buffer (pH 6.0). The reagrnt/AaHI molar ratio used varied from 2 to 40. After 30 min reaction at 20°C, an aliquot of the modified AaHI solution was withdrawn for determination of caseinolytic activity and hemorrhagic activity, respectively . The number of tryptophan residues oxidized was determined by amino acid analysis . Circubr dichroism andfluorescence spectra. CD spectra were measured with aJasco J-500 dichropohuimeter (Japan). The light path of the couches used was 0.3-0 .05 cm . All the data are expressed as molar residual ellipticity. The mean residue weight of AaHI was takrn as 110. The fluorescence spectrum of AaHI was measured with a Shimadzu RF Spectrofluorometer (Japan). The wavelength of excitation was 295 nm . The fluorescence intrnsitiea of AaHI were expressed as relative fluorescence intensity. Amino acid analysis . Analysis was performed with an automatic amino acid analyzer (Hitachi model 833 analyzer). Protein hydrolysis were carried out in 5.7 N HCl at 110°C under nitrogrn for 24 hr . Tryptophan was determined after hydrolysis with 4N p.tolurnesulphonic acid (Ltu and CtaAtvo, 1971). Enryme assay and analytical methods. Caseinolytic activity was measured colorimetrically according to the method of MIJaAi'A et al. (1%3). Hemorrhagic activity was determined by the method of Koxoo et al. (1960) . Immunoprxipitation was conducted in 1~ aqueous agar by the Ouchterlony method of double diffusion with antitoxic horse serum. Protein concentration was determined spectrophotometrically, assuming an absorbante of 1 .2 for 0.1% AaHI at 280 nm . RESULTS
Mod cation with DEP
The effects of various concentrations of DEP on enzyme activity are shown in Fig. 1 . The progress of oxidation was followed by measuring the change in absorbency at 240 nm (Fig. 1). The extent of modification of His residues was calculated from the increase in absorbance at 240 nm of the reaction solution (A~=3.2 x 10' M- ' cm - ') (MILES, 1977). The correlation between the degree of AaHI inactivation and the number of His modified by DEP is shown in Fig. 2. The result of amino acid analysis (data not shown) was consistent with that from spectrometric method . The results indicated that when approximately three residues of His per molecule of enzyme reacted with DEP, AaHI lost 85% of its original activity, not only its caseinolytic activity, but also its hemorrhegic and immunochemical activities (data not shown) . Most of the activity lost could be recovered upon treatment of the modified enzyme with NHZOH. Incubation in the absence of NH20H did not cause any restoration of activity .
Chemical Modification of AaHI
28 5
100 0.30
eo
0.20 {+
U O
y tr
40
0 aN
0 .10
0
0 1 10
I 0
I 20
I 30
(mmole)
DEP
I 40
Flo. 1 . REACTION OF DIETHYL PYROCARBONATE (DEP) wITH HEMORRHAOgc roxnv I (AaHI) . AaHI (2 .5 x 10-' M) was incubated with the indicated concentration of DEP at 20°C . The histidine reaction was revenged for 30 min by treatment with 0.4 M NH~OH (pH 7.0) at 5°C for 12 hr . " A increase in absorbance at 240 nm ; Q - _ _ _ _ _ O - " Enzyme activity; A absorbance at 240 nm of modified AaHI after treatment with NH~OH at pH 7.0.
Circular dichroism and fluorescence were also used to investigate the conformational aspects of modified AaHI. The CD spectra of DEP-inactivated AaHI and native AaHI are quite similar in the far u.v. region (Fig. 3). The maximum of the emission spectrum of AaHI was around 350 nm, and after AaHI was inactivated by DEP the emission maximum was still 350 nm, with a 30% diminution of intensity (Fig. 4). This indicated that only minor changes in tertiary structure had occurred . Since modification of Trp by DEP causes complete loss of fluorescence (ROSEN et al., 1970), it was apparent that not more than one Trp could have been modified by DEP. Inhibition of AaHl by NBS The effect of various concentration of NBS on caseinolytic activity is shown in Table 1 . A 30 fold molar excess of NBS over AaHI decreased the proteolytic activity by 80%. A similar result was observed for the hemorrhagic activity (data not shown) . Of the original activity, SO% remained after modification of all three tryptophan residues (Table 1). Although NBS has been shown to modify Trp residues in protein with considerable specificity ($PANDE et al., 1966). Tyrosine, histidine and methionine can also react with loor.\
?~ eo r
*~ so o p0
v
i
0 20 0
I 0
I I
I 2
\I 3
HIS residues modified / mol
FIa . 2. CORRELATION HETWF.EN INACTIVATION OF AaHI AND THE NUMBER OF HIS RESIDUES MODIFIED . AaHI (5 x 10 -' M) in 0.1 M acetate buffer (pH 6.0) was treated with various concentration of DEP at 20°C for 60 min. The number of His residues modified was calculated as described in the text, according to the method of SFANDE and WTTKOF (197n.
I 210
I 220
a
I 230 lnm)
I 240
I 250
AaHI . Native AaHI (0 .1 mg/ml) in 0.01 M acetate buffer (pH 6.0); - - - - - - AaHI plus 30 mM DEP after 30 min at 20°C; . . . . . . . . . . AaHI plus N-bromosuccimide (NBS/AaHI=30/1) after 30 min at 20°C . FIG . 3 . CIRCULAR DICHROISM SPECTRA OF
NBS (SHALTIEL and PATCHORNIK, 1963), especially at pH 6.6. The numbers of these amino acids from AaHI at various stages of oxidation are shown in Table 1 . Tyr and His decreased gradually during the treatment with NBS. Modification of AaHI with HNB-bromide, H~O~-dioxane, acetylimidazole and TNM Upon incubation of AaHI for 30 min with HNB-bromide (60 mM - 240 mM), Trp became modified ; 240 mM reagent resulted in the modification of 3 Trp residues, as determined both spectrophotometrically and by amino acid analysis . No loss of enzyme activity was observed . loo T N C N C N U C
ar " +
0
m
300
350
a
400
450
lnm)
FIG . 4 . FLUORESCENCE EMISSION SPECTRA OF ABHI . Native AaHI (3 x 10 -" M) in 0.01 M acetate buffer (pH 6.0); - - - - - - AaHI plus 30 mM DEP after 30 min at 20°C ; . . . . . . . . AaHI plus NBS (NBS/AaHI=30/1) after 30 min at 20°C . The fluorescence intensities of AaHI are expressed as relative fluorescence intensity. Excitation wavelength 295 nm .
Chemical Modification of AaHI
28 7
TABLE 1 . EFFECT OF OXIDATION OF ABHI (HEMORRFIAGIC TOXIN I) BY NBS (N BROMOS000IMIDE) ON ENZYMATIC ACTIVITY AND ON NUMBERS OF REACTIVE AMINO ACID RESIDUES
Molar excess
Amino acid "
Trpt Tyr Met His enzyme activity (%)*
NBS
0
4
l2
IS
20
30
3.2 9 4 7
2 .3 8 4 7
0.64 6 4 6
0 4 4 5
0 3 3.7 4
0 0 4 3.5
100
100
60
60
28
23
"The composition of other amino acids is not shown in the table for simplicity, as they were the same as those of the native enzyme . Trp was determined after hydrolysis by 4 N p-toluenesulphonic acid for 24 hr . *The actual absolute 100% value is 48 units. (The unit is defined as Ng tyrosine equivalent of TCA soluble product formed per min per mg protein.)
The result of oxidation of AaHI by H20z - dioxane was similar to that of HNB modification . Oxidation of AaHI was carried out with various concentrations of H202 -dioxane at pH 8.4 in 0.5 M NaHCO, buffer for 2.5 hr at 5°C. After incubation with 100 mM and 2fW mM HZOZ -dioxane 1 .13 and 2.6 out of 3 Trp residues were modified, respectively, but no change in enzyme activity was observed . Incubation of AaHI sodium barbital buffer, pH 7.5 for 30 min at 4°C in a pH-stat with a 200-fold molar excess of acetylimidazole resulted in the modification of 3 Tyr residues out of 9 present in AaHI . A S% loss of enzyme activity was observed, therefore modification of three Tyr residues in AaHI does not seem to influence its enzyme activity . The reaction of AaHI with TNM caused no loss of activity . DISCUSSION
Diethyl pyrocarbonate (DEP) is used for modification of the imidazole group. In the presence of 30 mM DEP, the enzyme activity decreased by 80%, accompanied by modification of 2 - 3 His residues out of the 7 present in Aa.HI. The loss of activity can be reversed by removing the carbethoxy group from the modified imidazole. Circular dichroism and fluorescence studies indicated that the modified enzyme retained its native conformation . Therefore, it could be deduced that the loss of the activity might be directly correlated with the modification of the His side chans). The extrapolated data shown in Fig. 2 indicated that'modification of three His residues abolished the activity of AaHI . It seems unlikely, however, that all three His residues could be involved in the active site of a protease . Considering that in a protein molecule some side chains have the same chemical potential to be modified, we can only deduce that at least one His residue is involved in the active site of the AaHI molecule . A gradual loss of activity was observed when AaHI was treated with NBS, which is a specific reagent for the modification of the Trp side chain at pH 4.5. It was found, however, that under the conditions used here, Tyr and His side chains of AaHI were modified simultaneously. In order to correlate the function of tryptophan side chains with the activity of AaHI, more evidence was necessary. The results given demonstrate that AaHI remains fully active upon oxidation of 2.6 Trp residues with HZOZ - dioxane. This
288
XUN XU
et al.
suggests that the side chains of Trp of AaHI are not essential for function . That HNBbromide treatment of AaHI did not damage the activity, supports this conclusion . It seems likely that Tyr side chains are also non-essential for the activity of AaHI, since the modification of Tyr with acetylimidazole did not affect AaHI activity . The finding that the treatment of AaHI with TNM had no effect on its activity supports this suggestion. We therefore conclude that one or two His residues) are involved in the active site of AaHI, while Trp and the reactive Tyr side chains are not essential for its activity . The modification of the essential His side chain may account for the loss of activity observed upon treatment with NBS, however, the active center of AaHI requires further study. Acknowledgment - we
thank Dr Xu GENG Jux for the gift of TNM. REFERENCES
B~tttitnrr, T. E. and Kosat.nxn, D. E. (1%7) A colorimetric procedure for the quantitatvn determination of tryptophan residues in protein. J. biol . Chem . 242, 5771 . B~Nex~sox, J. B. and Tu, A. T. (1978) Hemorrhagic toxins from western diamondback rattlesnake (Crotalus atrox) venom: isolation and characterization of five toxins and the role of zinc in hemorrhagic toxin e. Biochemistry 17, 3395 . HACHIMORI, Y., Hoatxtsxt, L, Kutttttnaw, K. and $HIBATA, K. (1964) Studies of amino acid residues in proteins . V. Different reactivities with H,O, of tryptophan residues in lysozyme, proteins and zymogens . Biochim. biophys. Acta 93, 346. Koxno, H., Koxno, S., IKezAWn, H., MURATA, K. and GHSnxn, A. (1960) Studies on the quantitative methods for determination of hemorrhagic activity of habu snake venom. Jap. J. med. Sci. Biol. 13, 43 . Liu, T. Y. and Cttvvo, Y. H. (1971) Hydrolysis of protein with p-toluenesulfonic acid determination of tryptophan . J. biol. Chem . 246, 2842 . M~1.ES, E. W. (1977) Modification of histidyl residues in proteins by diethylpyrocarbonate In : Methods in Enzymology, Vol. 47, 431 (H~xs, C. H. W. and TIMnsHEeF, S. N., Eds) . New York : Academic Press. MURATA, Y., SATAKE, M. and Suzuxt, T. (1%3) Studies on snake venom . XII. Distribution of proteinase activities among Japanese and Formosan snake venom. J. Biochem. 53, 431 R~ottn~+rt, J. F. and V.~t .LEe, B. C. (1972) 0 .acetyltyrosine . In : Methods in Enzymology, Vol. 25, p. 500 (H~xs, C. H. W. and TtMASHEFF, S. N., Eds) New York : Academic Press. RIORDAN, J. F. and VAL.LEE, B. C. (1972) Nitration with tetranitromethane. In : Methods in Enyymology, Vol. 25, p 515 (H~xs, C . H . W. and TtMast-~r-~, S. N., Eds) New York : Academic Press. Rosew, C. G., GEVALL, T. and AxnExssox, L. O. (1970) Reaction of diethylpyrocarbonate with indole derivatives with special reference to the reaction with tryptophan residues in a protein. Biochim. biophys. Acta 221, 207. SHALTIEL, S. and PALTCHORNIK, A. (1%3) Cleavage of histidyl peptide bonds by N-bromosuccinimide. J. Am . chem. Soc. 85, 2799 . SPANDE, T. F., GREEx, N. M. and WITKOI', B. (1966) The reactivity toward N-bromosuccinimide of tryptophan in enzymes, zymogens and inhibited enzymes. Biochemistry S, 1926. Tu, A. T. (1982) Hemorrhage induced by snake vcnoms . J. Formosan med. Ass. 81, 807. Xu, X., Wntvo, C. Ltu, J. and Lu, Z. (1981) Purification and characterization of hemorrhagic components from Agkistrodon acutus (hundred pace snake) venom Toxicon 19, 633 .