Toxicon VoL 30, No . 2, pp. I51-159, 1992. Printed in Otoat Britain .
0041-0101 f92 f5.00+ .00 ®1992 Perprttoa Ptew plc
IMMUNOCHEMICAL PROPERTIES OF NAJA NAJA ATRA (TAIWAN COBRA) PHOSPHOLIPASE AZ USING POLYCLONAL AND MONOCLONAL ANTIBODIES C. C. Ywxc, L. S.
CHwxc,
P. L. Oxc and T. H. Tuxc
Institute of Life Sciences, National Taing Hua University, Hsinchu, Taiwan 30043, Republic of China (Received 29 .Nay 1991 ; accepted 5 September 1991)
C. C. YANG, L. S. CI-I~xc, P. L. Oxc and T. H. Tvxc. Immunochemical properties of Naja raja atra (Taiwan cobra) phospholipase A2 using polyclonal and monoclonal antibodies. Toxicon 30, 151-159, 1992.-The immunochemical properties of Naja naja atra phospholipase A2 (NNA-PLAN were studied by using the chemically modified PLA2 derivatives and the PLA2 homologues toward anti-NNA-PLA2 polyclonal and monoclonal antibodies. Anti-NNA-PLA2 polyclonal antibodies inhibited the enzymatic activity of NNA-PLA2 and Hemachatus ihaemachatus DE-I by 87% and 68%, respectively . However, the enzymatic activities of Naja nigricollis CMS-9 and noteain were not significantly affected by the polyclonal antibodies. Competitive enzyme immunoassay revealed that the affinity of NNA-PLA2 for polyclonal antibodies was 330-fold higher than that of Hemacittatr~s i11aelllac%tattlS DE-I . Naja nigricollis CMS-9 and notexin failed to inhibit the binding of NNA-PLA 2 to polyclonal antibodies. This implies that the epitope(s) of NNA-PLA2 might comprise some substituted residues in the sequence of PLA2 homologues. Three monoclonal antibodies against NNA-PLA2 were prepared by a hybridoma technique. Two of these monoclonal antibodies inhibited the enzymatic activity of NNA-PLA2, but the other did not. Removal of the N-terminal octapeptide affected the epitope interacting with these monoclonal antibodies. Selective modification of tyrosine residues at positions 3 and 63 or lysine residues at positions 6 and 65 induced a substantial reduction in affinity of NNA-PLA2 for polyclonal and monoclonal antibodies. The three monoclonal antibodies failed to recognize PLA2 homologues. The comparison of the sequence of NNA-PLA2 to those of PLA2 homologues showed that most of the amino acid substitutions of PLA2 homologues occur in the spatially nearby region of the N-terminal region and residues at positions 63 and 65. These results suggest that anti-NNA-PLA 2 polyclonal antibodies and monoclonal antibodies are directed to epitope(s) composed of the N-terminal region, Tyr-63 and Lys-65 themselves and/or their neighbouring region . INTRODUCTION
A2 (PLA 2; EC 3.1 .1 .4), which specifically catalyses the hydrolysis of the fatty acid ester bond at position 2 of 1,2-diacyl-sn-phosphoglycerides, has been successfully purified from pancreatic tissues, and snake and bee venoms (Dsxxls, 1983; WITS, PH05PHOLIPASE
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198 . X-ray crystallographic analysis has provided models for the three-dimensional structure of PLA2 enzymes (DUKSTRA et al., 1981 ; BRUME et al., ]985; Wl~ et al., 1990) . The sequence homology and common mode of action of PLA2 enzymes imply that all PLA2 enzymes possess a similar overall structure, but differ in details such as the extent of secondary structure and positioning of an invariant side-chain (DIJI{STRA et al., 1983; RENE'ISEDER et al., 1985; TiFIUlvrnssEx et al., 1990). PLA2 from Naja raja atra snake venom is an acidic single chain polypeptide consisting of 120 amino acid residues with seven disulphide bonds (TSAI et al., 1981) . Its enzymatic and pharmacological properties have been reported (CONDREA et al., 1981a,b ; FLgrcIIER et al., 1981 ; RosExB>ntG et al., 1983) ; and its structure-function relationships have also been studied (YANG et al., 1981, 1982, 1985; YAxa and CHANG, 1984, 1988, 1989). However, the immunological properties of N. raja airs PLA2 (NNA-PLA2) remain to be resolved . In this study, polyclonal antibodies (pAbs) and monoclonal antibodies (mAbs) raised against NNA-PLA 2 were used to characterize its immunochemical properties. Chemically modified PLA 2 derivatives and structurally homologous variants of NNA-PLA 2 were used to examine the effect of modified and substituted residues on the binding of PLA2 with antibodies. Based on these results, the chemical structure of the epitope(s) in NNA-PLA 2 is discussed . Although synthetic peptides and anti-peptide antibodies are widely used in determining the sequential epitopes of protein (LERxER, 1982; ATASSI, 1984), the conformational dependent epitopes were not effectively elucidated by this method . We found that studies on the differential binding affinity of anti-NNA-PLA 2 antibodies to NNA-PLA2 , chemically modified PLA2 derivatives and PLA2 homologues could provide an approach to elucidate the conformational dependent epitope(s) of NNA-PLA 2 .
MATERIALS AND METHODS Materials PLA2 enzymes from N. nafa atra (Taiwan cobra), N. nigricollis (spitting cobra) and Hemachatus hacmachatus (Ringhala) venoms, and notexin from NotechLr scutatus scutatus (Australian tiger snake) venom were isolated and purified as previously described (Yexa and ICnva, 1980a,b; Yetvc et al., 1981 ; Y~rta and Ct~xa, 1990) . The purity of PLA2 enzymes and notexin was verified by polyacrylamide gel electrophoresis (PAGE). Hypoxanthine-atninopterin-thymidine (HAT) and hypoxanthine-thymidine (HT) selection media were purchased from Hazalton Biolo®ics, Inc. (Lenexa, ICS, U.S.A .) . Polyethyleneglycol (PEG) 1500 and 2,2= azino-di[3-ethylbenztlvazoGnesulphonic acid] (ABTS) were purchased from Boehringer Mannheim Gmbh, Biochetnica (D-6800 Mannheim 31, F.R.G.) . Sephacryl S-300 was obtained from Pharmacie LKB Biotechnology (IJppaala, Sweden) and 2,6,10,14tetramethylpentadecane (pristane) from Sigma Chemical Company (St. Louis, MO, U.S .A .) . Carrier-free Na'~I was purchased from NEN Research Products (Boston, MA, U.S .A .) and peroxidase-conjugated goat anti-rabbit IgG from Jackson Immunoresearch Laboratories, Inc. (West Grove, PA, U.S .A.) . All other reagents were of analytical grade.
Preparation ojpolyclorral antibodies against NNA-PLA 2 Rabbits weighing about 2.5 kg wets injected during a period of two months with increasing doses of NNA-PLA2 (from 100 ug to 2.6 mg/kg body weight) emulsified with an equal volume of Freund's complete adjuvant (Sigma Chemical Company). The rabbits were bled 9 days after the final immunization. Antisera were precipitated with 50% saturated ammonium sulphate. The precipitates were dialysed exhaustively against 0.02 M Tris-0.15 M NaCI (pH 7.5). This antibody preparation was used for determination of the inhibitory capacity of polyclonal antibodies (pAbs) on the PLA2 activities of PLA2 enzymes and notexin. Protein concentration was estimated according to the equation, A2w (27+AZSVlA 2~ = protein concentration (in mg/ml) (EIwacJow and LANE, 1988). Am, and A2~ were the absorbance at 205 nm and 280 nm, respectively .
Naja naja afro Phospholipase Ai
153
Preparation ojmonoelonal antibodies against NNA-PLA Z Monoclonal antibodies (mAbs) were produced by a modification of the method of KBHt.rrt and MustEix (1975). Balb/c mice were immunised with NNA-PLAZ (10, 20, 30, 50, 70, 100 pgJmouse) s.c . at 2-week intervals. An additional boost was injected 3 days before fusion . Spleen cells wen fused with myeloma cells, NS-1 cells, using PEG 1500 . After removal of PEG 1500, cells were suspended in a HAT selection medium and cultured with .HAT and HT selection media until disappearance of myeloma cells. Hybrid-containing wells wen tested by enzyme immunoassay (JueNa et al., 1981) for anti-PLA Z antibody . Positive clones were subcloned by limiting dilution . Hybridoma cells producing mAbs wen injected i.p. into pristane-primed Balb/c mice. Ascites fluid was withdrawn and purified by amity chromatography on a PLAZ-Scpharose 4B column as described previously (Y~tva et al., 1977). The purity of the mAbs was verified by SDS-PAGE . Three mAbs producing clones (1E531, SF92 and SF6F10) wen obtained and their subclasses were determined by indirect enzyme immunoassay using the SBA clonotyping system III kit (Southern Biotechnology Associates, Inc., Birmingham, AL, U.S.A .). The isotype of 1E531 and SF92 is IgG,(x), and SF6F10 belongs to IgGZ, (x). The dissociation constants of mAbs with NNA-PLAZ wen measured from the radioimmunoassay binding experiments by Scatchard plot analysis. The dissociation constants of PLA Z with 1E531, SF92 and SF6F10 were 3.05 x 10 -° M, 2.76 x 10 -' M and 3.68 x l0 -" M, respectively . Preparation of clumically modified NNA-PLA s derivatives PLAZ was modified with p-bromophenacyl bromide (BPB-Br), and the only incorporated BPB group was found on His~7 (Y~a et al., 1981). Tyrosine and lysine modified derivatives wen prepared as described by Yexa et a1. (1985) and Y~rtG and G~Na (1989). The tyrosine-modified derivative, NBS-2, was selectively modified at Tyr~3, and NBS-4 at Tyr-3 and Tyr-63 . Only Lys-6 was modified in TNP-1, while both Lys-6 and Lys-65 wen modified in TNP-2. NNA-PLAZ was cleaved with CNBr as previously described (Yt xa and Cruxc, 1988), and the N-terminal octapeptide (CBI) was separated from the large C-terminal fragment (CBII) by high performance liquid chromatography . Reduction and S-carbozymethylation of NNA-PLA Z was performed according to the method of Cxssrt~t .n et al. (1963) . Competitive enzynrc immunoassay (EIA) EIA polystyrene microtitration wells were coated with 50 pl of 3.57 nM NNA-PLA Z and incubated at 4°C overnight. The plate was washod three times with phosphate buffered saline-Tween 20 (PBST). Auer the uncoated apace was blocked with I00 kl of 5% non-fat milk in PHS at 37°C for 2 hr, the plate was washod three times with PBST. The proper dilution of anti-NNA-PLAZ antisera (8000 x) was preincubated with PLAZ enzymes or modified derivatives at 37°C for 3 hr and at 4°C for 1 hr, then the incubation mixtures were added into the plate and incubated at 37°C for 3 hr and 4°C for I hr. After washing three times with PBST, 50 pl of peroxidase-conjugated goat anti-rabbit IgG (5000 x) in PBS was added. Following five washings, 50 pl of freshly prepared substrate solution (L5 mg ABTS in 10 ml of 0.1 M citrate buffer, pH 4.2, containing 0.009% HZOZ) was added. After incubation at 37°C for 8 min, the absorbance at 405 nm was measured . Competitive radioimmiaeoauay (RIA) Iodination of NNA-PLAZ was performed according to the method described by Ho~tvx and Gr~woon (1962) . Competitive RIA was carried out according to the method of Ci.EZAADnv et al. (1986) with a alight modification . RIA plate was coated with 50 pl of 6.6 nM mAb and incubated at 4°C overnight. After the uncoated space was blocked, 50 pl of the unlabelled PLAZ enzymes or modified derivatives in serial dilutions wen added and incubated at 37°C for 3 hr, then at 4°C for 1 hr . Finally, 50 pl of '~`I-labelled NNA-PLAZ (4 x 10' cpm/ml) was added. Radioactivity of the plate was measured in a gamma counter. The ratio of the bound radiohibelled antigen in the presence of inhibitor to that in the absence of inhibitor was plotted against the concentration of the inhibitor added. The concentration of the inhibitor which gave 50% inhibition (IC,o) was determined. RESULTS
As seen in Fig. 1, the enzymatic activity of NNA-PLAZ was inhibited with increasing amounts of anti-PLAZ pAbs. The pAbs also reduced the enzymatic activity of H. haemachatus DirI by 68% . However, the enzymatic activities of N. nigricollis CMS-9 and notexin were not affected significantly .
154
C . C . YANG et al.
100 T
Gi 4 ca E N C W
ao
FYc. 1 . IxFnemoty oP PLA= ecrIVITV of PLA2 srlzv~s ANU NoTEImv By eNn-NNA-PLA2 pAbs. Variable amounts of pAbs were preincubated with 2 kg NNA-PLA= (p), H. haemachatus D)rI (p), N. nigricollir CMS-9 (p) and notexin (p) at 37°C for 30 min . The remaining PLAZ activity of the respective enzymes was related to the activity in the absence of anti-NNA-PLA z pAbs . The absolute 100% PLA2 activity of NNA-PLAT H. haemachatur D)rI, N. nigricollis CMS-9 and notexin was 1800, 900, 420 and 980 Nequiv of free fatty acids liberated per min per mg, respectively. Determination of enzymatic activity was performed in the same way as previously described (Yelvc and KING, 1980a) . All values are the means of triplicate determinations.
100
ao
10
10 °
10
-s
~
10~
C~s'etitsr (pM x 3 .571 FIC . 2 . INHIBITION
To ~Nn-NNA-PLA 2 pAbs By PLAN sNZVxus exn NHS-4 . The detailod procedure was described in the Materials and Methods section . AJAo was determined as the ratio (%) of absorbance at 405 nm in the presence (A) and absence of competitor (Ao) . The symbols Q, Q, pand ~ represented NNA-PLA2, H. haemachatus D)ïI, N. nigricollis CMS-9 and NBS-4, respectively. Notexin aad N. nigricollls CMS-9 failed to inhibit the binding of NNA-PLAZ to Abs. All values are the means of triplicate determinations. OF
NNA-PLA2 Bnvnuvc
Naja naja arra Phospholipase AZ
15 5
roo U Q ti ~O
E
60
N C W
0
0 .6
Môlar Ratio of mAb to PLAZ
1
Frc. 3. Ir~arnoN oP exzYwunc ecnvrrsr of NNA-PLAZ aY mAbs . NNA-PLAZ wes preincubatod with variable amounts of mAbs, 1E531 (~), SF92 (~) and SF6F10 (/) at 37°C for 2 hr before being assayed . All values are the means of triplicate determinations .
The amino acid sequence of NNA-PLAZ showed a 85%, 77% and 53% sequence homology with that of H. haemachatus DE-I, N. nigricollis CMS-9 and notexin, respectively (ICnaI and IWAxAGA, 1986). Competitive EIA revealed that NNA-PLAZ had an Abs binding affinity 330-fold higher than H. haemachatus DE-I (Fig. 2). This implies that the substituted residues appearing in H. haemachatus DE-I might be related to the epitope(s) of NNA-PLAZ. Further amino acid substitutions occumng in N. nigricollis CMS-9 and noteatin caused them to fail to inhibit the binding of NNA-PLAZ to anti-PLA Z pAbs. Modifications of Tyr-3 and Tyr-63 of NNA-PLAZ (NBS-4) caused some reduction in its binding affinity for pAbs (Fig. 2). Modified derivatives, NBS-2 (modified on Tyr-63), TNP-1 (modified on Lys-6) and TNP-2 (modified on Lys-6 and Lys-65) as well as NBS-4 also exhibited lower Abs binding affinities than the native enzyme (data not shown) . This suggests that Tyr-3, Lys-6, Tyr-63 and Lys-65 might be located on or in proximity to the epitope(s) of NNA-PLAZ. Although the three mAbs (1E531, SF92 and SF6F10) all bound with NNA-PLAZ, the enzymatic activity of NNA-PLAZ was only inhibited by 1E531 and SF92 (Fig. 3). At a molar ratio of l :l, 78% and 90% inhibition for 1E531 and SF92 were observed. However, SF6F10 did not abolish the PLAZ activity. This indicated that the epitope recognized by SF6F10 might be topographically different from that recognized by 1E531 and SF92 . The molar ratios of Ag : Ab of Ag-mAbs complexes which appeared in the incubation mixtures of NNA-PLAZ with two or three kinds of mAbs, calculated from their apparent molecular weights by gel filtration on Sephacryl S-300 column, were 1 :1 (data not shown) . This finding suggests that these mAbs do not bind simultaneously with NNA-PLAZ, and that the epitopes of PLAZ recognized by these three mAbs are located on close or partially overlapping regions. The failure of these three mAbs to react with reduced and S-carboxymethylated NNA-PLAZ indicated that the epitopes recognized by these mAbs are conformational dependent ones . As shown in Table 1, BPB-PLAZ displayed a slight decrease in ai$nities for the three mAbs . Selective modification of tyrosine residues (at positions 3 and 63) and lysine residues (at positions 6 and 65) of NNA-PLAZ all induced a marked reduction in affinities for mAbs . In view of the changes in their lei values, it would seem that lysine residues
156
C. C. YANG et al.
played a more crucial role in the interaction of mAbs with NNA-PLAZ than tyrosine residues . After removal of the N-terminal octapeptide, the Icy value drastically increased. These results suggest that the N-terminal region and the modified residues might belong to the epitope recognized by these mAbs. Alternatively, the inability of PLAZ homologues to compete with NNA-PLAZ for binding mAbs implies that some of the substituted residues in PLAZ homologues are involved in the Ag-mAb interaction. DISCUSSION
In this study, the derivatives of NNA-PLAZ modified either at tyrosine or lysine residues showed a decrease in affinities for anti-PLAZ pAbs and mAbs as compared with native enzyme (Table 1). Removal of the N-terminal octapeptide also affected the epitopes interacting with the mAbs. Our previous finding (YANG and CHANG, 1988) using anti-PLAZ pAbs proved that the N-terminal region is one of epitopes in NNA-PLAZ. These results suggest that the N-terminal region, Tyr-63 and Lys-65 might play a critical role in the Ag-Ab interaction. Although these residues are separated in the peptide sequence, the three-dimensional structure of NNA-PLAZ (WHITE et al., 1990 ; ScoTT et al ., 1990) shows that these residues are brought together by virtue of folding of the polypeptide chain (Fig. 4). Thus, it is likely that these residues themselves and/or their nearby region mutually construct the conformational dependent epitope(s) of NNA-PLAZ. The comparison of the amino acid sequence of NNA-PLAZ to those of PLAZ homologues shows that the greater part of substituted residues occur in the spatial region surrounding the N-terminal region, and residues at homologous positions 63 and 65. The fact that the homologous variants of NNA-PLAZ had a lower reactivity toward anti-PLAZ pAbs and mAbs supports the view that the epitope(s) of NNA-PLAZ might be composed of the N-terminal region, Tyr-63 and Lys-65 and/or their neighbouring regions. Prediction of the probable epitope regions from the hydrophilicity profile (Hoof and WOODS, 1981) TAeLe 1. INHHt1170N oe 'vI-r .na~ .rF NNA-PLAz anvDnva ro mAbs sY CHH~CALLY MODQ+~.D NNA-PLAZ DFRIVATIYES AND PLAz HOMOLOCiUP3
1E531 Native NNA-PLAZ Modified NNA-PLA=: BPB (His-47)' TNP-1 (Lys-6) TNP-2 (Lys-6 & 65) NBS-2 (Tyr-63) NBS-4 (Tyr-3 dc 63) CBIIt PLAZ homologuea : X. haemachatw DE-I N. nigricollis CMS-9 Notexin
(IIM) SF92
IC~p
SF6F10
105
46
45
228 1570 2860 577 741 3409
113 970 2120 386 970 2654
67 1456 2588 427 1966 6062
> 10'
S350 8606 > 10~
6535 5912 > 10`
> 10' > 10'
The numbers in parentheses indicate the positions of modified residues in NNA-PLA=. t NNA-PLAN was cleaved with CNBr, and the N-terminal octapeptide (CBn was separated from the large C-terminal fragment (CBII) (YANO and CFIANG, 1988).
Naja naja atra PhosphoGpase A2
157
Fyc . 4. TERTIARY STRUCTURE OF NNA-PLAZ sxownvc T~ REUrtvE r oceTCOxs of T~ Monu~o
RP3II)UFS. The model is based on the result of Sara et al. (1990) . Numbers in parentheses indicate the positions of Tyr-3, Lys-6, Tyr-63 and Lys-65 in NNA-PLAN.
t â 0 ôT 2
2 -4 v
20
40
60
~
80
v
100
o 120
Sequence position Flc. 5. HYDROPATHY PROtIIL Or NNA-PLA2. The averaged hydropathic indexes were plotted vs the amino acid sequence of NNA-PLAN according to the method of Hour and Woons (1981). The x-axis contains l20 increments, each representing an amino acid in the sequence of NNA-PLA2. The y-axis represents the hydropathic indexes represented as the averages of seven residues . The data points are plotted at the centre of the averaging group from which they were derived. The bar represents the probable epitope regions (floor and Woous, 1981).
158
C . C. YANG er a/.
of the amino sequence of NNA-PLAZ also suggests that segments 1-7 and 62-72 are two of the five candidate epitope regions shown in Fig. 5. The residues of the N-terminal region, Tyr-63 and Lys-65 are conserved residues in NNA-PLA Z and H. haemachatus DE-I (Klxl and Iwwxwcw, 1986); however, competitive RIA revealed that H. haemachatus DE-I has a weak affinity for anti-NNA-PLA Z mAbs . This implies the involvement of other residues in the interaction between mAbs and NNA-PLA Z besides the N-terminal region, Tyr-63 and Lys-65 . In view of the inhibitory capacities of mAbs on PLA Z activity, it is plausible that the other residues of the epitopes recognized by 1E531 and SF92 were neighbouring on or were part of the catalytic site . Consequently, the enzymatic activity of NNA-PLAZ was inhibited. The inability of SF6F10 to inhibit the PLAZ activity suggests that the other residues in its epitope were topographically distant from the catalytic site of NNA-PLAZ . Acknowledgements`This work was supported by the National Science Council, Republic of China . We thank Dr J. Teusoo'r-r' for reading this manuscript, and also Miss F. S . Wu for purification of PLAz enzymes and notexin .
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LraxEa, R. A . (1982) Tapping the immunological repertoire to produce antibodies of predetermined specificity. Nature 299, 592-5% . RsNStseoea, R ., Bxutvn:, S ., Dutcsran, B. W ., DR~vrH, J . and S~ct~x, P. H. (1985) A comparison of the crystal structures of phoapholipase Az from bovine pancreas and Crotales atrox venom . J. biol. Chem . 260, 11,627-11,634. RostareEac, P ., Corroxa+, E., R~univo, B . E ., $GONS, K. R. and Y~rc, C . C. (1983) Dissociation of pharmacological and enzymatic activities of snake venom phospholipases AZ by modification of carboxylate groups . Biochem. Pharmac . 32, 3524-3530 . Scow, D. L., WxiTe, S. P ., OrwiNOwsict, Z ., Yuert, W., GEt .e, M . H . and S~ct ete, P. B . (1990) Interfacial catalysis: the mechanism of phospholipase A2 . Science 250, 1541-1546 . Txuxxts~v, M . M . G . M ., Kwc.tc, K. H ., DxaNrtt, J . and Dutcsrge, B. W . (1990) Structure of an engineered porcine phospholipase AZ with enhanced activity at 2.1 A tnsolution. J. molec . Biol. 216, 42539. Tsnt, I . H ., Wu, S. H. and Lo, T. B . (1981) Complete amino acid sequence of a phospholipase A= from the venom of Naja naja afro (Taiwan cobra) . Toxicon 19, 141-152. W~, M. (1987) Phospholipases, pp . 155-241 . New York : Plenum Press . Wtit~, S . P., Scow, D . L ., t7rwnvowsgt, Z ., Grt,a, M . H . and Stat-©e, P. B . (1990) Crystal structure of cobra venom phospholipase A2 in a complex with transition-state analogue. Science 250, 1560-1563 . YANG, C. C . and Crnwe, L. S . (1984) Tryptophan modification of phosopholipase AZ enzymes and presynaptic neurotoxins from snake venom . J. protein Chem. 3, 195-213 . YeNC, C . C. and CtiANG, L . S. (1988) Role of the N-terminal region in phospholipase Az from Naja naja afro (Taiwan cobra) and Naja nigricollis (spitting cobra) venons . Toxicon 26, 721-731 . YANG, C . C . and C~NC, L. S. (1989) Studies on the status of lysine residues in phospholipase AZ from Naja nçja afro (Taiwan cobra) snake venom . Biochem. J. 262, 855-860. YnNC, C . C . and C~uNC, L. S . (1990) The N-terminal amino group essential for the biological activity of notexin from Notechis scutatus seatatus venom . Biochim . biophys. Acta 1040, 351 . YANG, C. C . and K~NC, K . (1980x) Chemical modification of the histidine residue in basic phospholipase A~ from Naja nigricollis snake venom. Biochim . biophys. Acta 614, 373-388 . YANG, C . C . and K~NC, K . (19806) Chemical modification of the histidine residue in phoapholipase Az from the Hemachatur haemachatus snake venom . Toxicon 18, 529-547. Yertc, C . C ., L1N, M . F . and CtteNC, C . C . (1977) Purification of anti-cobrotoxin antibody by affinity chromatography . Toxicon 15, 512. Y~rrG, C. C ., Ktxc, K . and Sex, T . P. (1981) Chemical modification of lysine and histidine residues in phospholipase AZ from the venom of Naja naja afro (Taiwan cobra) . Toxicon 19, 645-659. YANG, C . C ., KrNC, K., Sex, T. P. and Hseu, W . S . (1982) Lysine modification in snake venom phospholipases A2 . The Snake 14, 110-118 . Yexc, C . C ., HUANG, C . S . and Lam, H . J . (1985) Studies on the status of tyrosyl residues in the phospholipase Az from Naja naja afro and Naja nigricollis snake venons . J. protein Chem . 4, 87-102 .
0041-0101 f92 f5.00+ .00 ®1992 Perprttoa Ptew plc
IMMUNOCHEMICAL PROPERTIES OF NAJA NAJA ATRA (TAIWAN COBRA) PHOSPHOLIPASE AZ USING POLYCLONAL AND MONOCLONAL ANTIBODIES C. C. Ywxc, L. S.
CHwxc,
P. L. Oxc and T. H. Tuxc
Institute of Life Sciences, National Taing Hua University, Hsinchu, Taiwan 30043, Republic of China (Received 29 .Nay 1991 ; accepted 5 September 1991)
C. C. YANG, L. S. CI-I~xc, P. L. Oxc and T. H. Tvxc. Immunochemical properties of Naja raja atra (Taiwan cobra) phospholipase A2 using polyclonal and monoclonal antibodies. Toxicon 30, 151-159, 1992.-The immunochemical properties of Naja naja atra phospholipase A2 (NNA-PLAN were studied by using the chemically modified PLA2 derivatives and the PLA2 homologues toward anti-NNA-PLA2 polyclonal and monoclonal antibodies. Anti-NNA-PLA2 polyclonal antibodies inhibited the enzymatic activity of NNA-PLA2 and Hemachatus ihaemachatus DE-I by 87% and 68%, respectively . However, the enzymatic activities of Naja nigricollis CMS-9 and noteain were not significantly affected by the polyclonal antibodies. Competitive enzyme immunoassay revealed that the affinity of NNA-PLA2 for polyclonal antibodies was 330-fold higher than that of Hemacittatr~s i11aelllac%tattlS DE-I . Naja nigricollis CMS-9 and notexin failed to inhibit the binding of NNA-PLA 2 to polyclonal antibodies. This implies that the epitope(s) of NNA-PLA2 might comprise some substituted residues in the sequence of PLA2 homologues. Three monoclonal antibodies against NNA-PLA2 were prepared by a hybridoma technique. Two of these monoclonal antibodies inhibited the enzymatic activity of NNA-PLA2, but the other did not. Removal of the N-terminal octapeptide affected the epitope interacting with these monoclonal antibodies. Selective modification of tyrosine residues at positions 3 and 63 or lysine residues at positions 6 and 65 induced a substantial reduction in affinity of NNA-PLA2 for polyclonal and monoclonal antibodies. The three monoclonal antibodies failed to recognize PLA2 homologues. The comparison of the sequence of NNA-PLA2 to those of PLA2 homologues showed that most of the amino acid substitutions of PLA2 homologues occur in the spatially nearby region of the N-terminal region and residues at positions 63 and 65. These results suggest that anti-NNA-PLA 2 polyclonal antibodies and monoclonal antibodies are directed to epitope(s) composed of the N-terminal region, Tyr-63 and Lys-65 themselves and/or their neighbouring region . INTRODUCTION
A2 (PLA 2; EC 3.1 .1 .4), which specifically catalyses the hydrolysis of the fatty acid ester bond at position 2 of 1,2-diacyl-sn-phosphoglycerides, has been successfully purified from pancreatic tissues, and snake and bee venoms (Dsxxls, 1983; WITS, PH05PHOLIPASE
152
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198 . X-ray crystallographic analysis has provided models for the three-dimensional structure of PLA2 enzymes (DUKSTRA et al., 1981 ; BRUME et al., ]985; Wl~ et al., 1990) . The sequence homology and common mode of action of PLA2 enzymes imply that all PLA2 enzymes possess a similar overall structure, but differ in details such as the extent of secondary structure and positioning of an invariant side-chain (DIJI{STRA et al., 1983; RENE'ISEDER et al., 1985; TiFIUlvrnssEx et al., 1990). PLA2 from Naja raja atra snake venom is an acidic single chain polypeptide consisting of 120 amino acid residues with seven disulphide bonds (TSAI et al., 1981) . Its enzymatic and pharmacological properties have been reported (CONDREA et al., 1981a,b ; FLgrcIIER et al., 1981 ; RosExB>ntG et al., 1983) ; and its structure-function relationships have also been studied (YANG et al., 1981, 1982, 1985; YAxa and CHANG, 1984, 1988, 1989). However, the immunological properties of N. raja airs PLA2 (NNA-PLA2) remain to be resolved . In this study, polyclonal antibodies (pAbs) and monoclonal antibodies (mAbs) raised against NNA-PLA 2 were used to characterize its immunochemical properties. Chemically modified PLA 2 derivatives and structurally homologous variants of NNA-PLA 2 were used to examine the effect of modified and substituted residues on the binding of PLA2 with antibodies. Based on these results, the chemical structure of the epitope(s) in NNA-PLA 2 is discussed . Although synthetic peptides and anti-peptide antibodies are widely used in determining the sequential epitopes of protein (LERxER, 1982; ATASSI, 1984), the conformational dependent epitopes were not effectively elucidated by this method . We found that studies on the differential binding affinity of anti-NNA-PLA 2 antibodies to NNA-PLA2 , chemically modified PLA2 derivatives and PLA2 homologues could provide an approach to elucidate the conformational dependent epitope(s) of NNA-PLA 2 .
MATERIALS AND METHODS Materials PLA2 enzymes from N. nafa atra (Taiwan cobra), N. nigricollis (spitting cobra) and Hemachatus hacmachatus (Ringhala) venoms, and notexin from NotechLr scutatus scutatus (Australian tiger snake) venom were isolated and purified as previously described (Yexa and ICnva, 1980a,b; Yetvc et al., 1981 ; Y~rta and Ct~xa, 1990) . The purity of PLA2 enzymes and notexin was verified by polyacrylamide gel electrophoresis (PAGE). Hypoxanthine-atninopterin-thymidine (HAT) and hypoxanthine-thymidine (HT) selection media were purchased from Hazalton Biolo®ics, Inc. (Lenexa, ICS, U.S.A .) . Polyethyleneglycol (PEG) 1500 and 2,2= azino-di[3-ethylbenztlvazoGnesulphonic acid] (ABTS) were purchased from Boehringer Mannheim Gmbh, Biochetnica (D-6800 Mannheim 31, F.R.G.) . Sephacryl S-300 was obtained from Pharmacie LKB Biotechnology (IJppaala, Sweden) and 2,6,10,14tetramethylpentadecane (pristane) from Sigma Chemical Company (St. Louis, MO, U.S .A .) . Carrier-free Na'~I was purchased from NEN Research Products (Boston, MA, U.S .A .) and peroxidase-conjugated goat anti-rabbit IgG from Jackson Immunoresearch Laboratories, Inc. (West Grove, PA, U.S .A.) . All other reagents were of analytical grade.
Preparation ojpolyclorral antibodies against NNA-PLA 2 Rabbits weighing about 2.5 kg wets injected during a period of two months with increasing doses of NNA-PLA2 (from 100 ug to 2.6 mg/kg body weight) emulsified with an equal volume of Freund's complete adjuvant (Sigma Chemical Company). The rabbits were bled 9 days after the final immunization. Antisera were precipitated with 50% saturated ammonium sulphate. The precipitates were dialysed exhaustively against 0.02 M Tris-0.15 M NaCI (pH 7.5). This antibody preparation was used for determination of the inhibitory capacity of polyclonal antibodies (pAbs) on the PLA2 activities of PLA2 enzymes and notexin. Protein concentration was estimated according to the equation, A2w (27+AZSVlA 2~ = protein concentration (in mg/ml) (EIwacJow and LANE, 1988). Am, and A2~ were the absorbance at 205 nm and 280 nm, respectively .
Naja naja afro Phospholipase Ai
153
Preparation ojmonoelonal antibodies against NNA-PLA Z Monoclonal antibodies (mAbs) were produced by a modification of the method of KBHt.rrt and MustEix (1975). Balb/c mice were immunised with NNA-PLAZ (10, 20, 30, 50, 70, 100 pgJmouse) s.c . at 2-week intervals. An additional boost was injected 3 days before fusion . Spleen cells wen fused with myeloma cells, NS-1 cells, using PEG 1500 . After removal of PEG 1500, cells were suspended in a HAT selection medium and cultured with .HAT and HT selection media until disappearance of myeloma cells. Hybrid-containing wells wen tested by enzyme immunoassay (JueNa et al., 1981) for anti-PLA Z antibody . Positive clones were subcloned by limiting dilution . Hybridoma cells producing mAbs wen injected i.p. into pristane-primed Balb/c mice. Ascites fluid was withdrawn and purified by amity chromatography on a PLAZ-Scpharose 4B column as described previously (Y~tva et al., 1977). The purity of the mAbs was verified by SDS-PAGE . Three mAbs producing clones (1E531, SF92 and SF6F10) wen obtained and their subclasses were determined by indirect enzyme immunoassay using the SBA clonotyping system III kit (Southern Biotechnology Associates, Inc., Birmingham, AL, U.S.A .). The isotype of 1E531 and SF92 is IgG,(x), and SF6F10 belongs to IgGZ, (x). The dissociation constants of mAbs with NNA-PLAZ wen measured from the radioimmunoassay binding experiments by Scatchard plot analysis. The dissociation constants of PLA Z with 1E531, SF92 and SF6F10 were 3.05 x 10 -° M, 2.76 x 10 -' M and 3.68 x l0 -" M, respectively . Preparation of clumically modified NNA-PLA s derivatives PLAZ was modified with p-bromophenacyl bromide (BPB-Br), and the only incorporated BPB group was found on His~7 (Y~a et al., 1981). Tyrosine and lysine modified derivatives wen prepared as described by Yexa et a1. (1985) and Y~rtG and G~Na (1989). The tyrosine-modified derivative, NBS-2, was selectively modified at Tyr~3, and NBS-4 at Tyr-3 and Tyr-63 . Only Lys-6 was modified in TNP-1, while both Lys-6 and Lys-65 wen modified in TNP-2. NNA-PLAZ was cleaved with CNBr as previously described (Yt xa and Cruxc, 1988), and the N-terminal octapeptide (CBI) was separated from the large C-terminal fragment (CBII) by high performance liquid chromatography . Reduction and S-carbozymethylation of NNA-PLA Z was performed according to the method of Cxssrt~t .n et al. (1963) . Competitive enzynrc immunoassay (EIA) EIA polystyrene microtitration wells were coated with 50 pl of 3.57 nM NNA-PLA Z and incubated at 4°C overnight. The plate was washod three times with phosphate buffered saline-Tween 20 (PBST). Auer the uncoated apace was blocked with I00 kl of 5% non-fat milk in PHS at 37°C for 2 hr, the plate was washod three times with PBST. The proper dilution of anti-NNA-PLAZ antisera (8000 x) was preincubated with PLAZ enzymes or modified derivatives at 37°C for 3 hr and at 4°C for 1 hr, then the incubation mixtures were added into the plate and incubated at 37°C for 3 hr and 4°C for I hr. After washing three times with PBST, 50 pl of peroxidase-conjugated goat anti-rabbit IgG (5000 x) in PBS was added. Following five washings, 50 pl of freshly prepared substrate solution (L5 mg ABTS in 10 ml of 0.1 M citrate buffer, pH 4.2, containing 0.009% HZOZ) was added. After incubation at 37°C for 8 min, the absorbance at 405 nm was measured . Competitive radioimmiaeoauay (RIA) Iodination of NNA-PLAZ was performed according to the method described by Ho~tvx and Gr~woon (1962) . Competitive RIA was carried out according to the method of Ci.EZAADnv et al. (1986) with a alight modification . RIA plate was coated with 50 pl of 6.6 nM mAb and incubated at 4°C overnight. After the uncoated space was blocked, 50 pl of the unlabelled PLAZ enzymes or modified derivatives in serial dilutions wen added and incubated at 37°C for 3 hr, then at 4°C for 1 hr . Finally, 50 pl of '~`I-labelled NNA-PLAZ (4 x 10' cpm/ml) was added. Radioactivity of the plate was measured in a gamma counter. The ratio of the bound radiohibelled antigen in the presence of inhibitor to that in the absence of inhibitor was plotted against the concentration of the inhibitor added. The concentration of the inhibitor which gave 50% inhibition (IC,o) was determined. RESULTS
As seen in Fig. 1, the enzymatic activity of NNA-PLAZ was inhibited with increasing amounts of anti-PLAZ pAbs. The pAbs also reduced the enzymatic activity of H. haemachatus DirI by 68% . However, the enzymatic activities of N. nigricollis CMS-9 and notexin were not affected significantly .
154
C . C . YANG et al.
100 T
Gi 4 ca E N C W
ao
FYc. 1 . IxFnemoty oP PLA= ecrIVITV of PLA2 srlzv~s ANU NoTEImv By eNn-NNA-PLA2 pAbs. Variable amounts of pAbs were preincubated with 2 kg NNA-PLA= (p), H. haemachatus D)rI (p), N. nigricollir CMS-9 (p) and notexin (p) at 37°C for 30 min . The remaining PLAZ activity of the respective enzymes was related to the activity in the absence of anti-NNA-PLA z pAbs . The absolute 100% PLA2 activity of NNA-PLAT H. haemachatur D)rI, N. nigricollis CMS-9 and notexin was 1800, 900, 420 and 980 Nequiv of free fatty acids liberated per min per mg, respectively. Determination of enzymatic activity was performed in the same way as previously described (Yelvc and KING, 1980a) . All values are the means of triplicate determinations.
100
ao
10
10 °
10
-s
~
10~
C~s'etitsr (pM x 3 .571 FIC . 2 . INHIBITION
To ~Nn-NNA-PLA 2 pAbs By PLAN sNZVxus exn NHS-4 . The detailod procedure was described in the Materials and Methods section . AJAo was determined as the ratio (%) of absorbance at 405 nm in the presence (A) and absence of competitor (Ao) . The symbols Q, Q, pand ~ represented NNA-PLA2, H. haemachatus D)ïI, N. nigricollis CMS-9 and NBS-4, respectively. Notexin aad N. nigricollls CMS-9 failed to inhibit the binding of NNA-PLAZ to Abs. All values are the means of triplicate determinations. OF
NNA-PLA2 Bnvnuvc
Naja naja arra Phospholipase AZ
15 5
roo U Q ti ~O
E
60
N C W
0
0 .6
Môlar Ratio of mAb to PLAZ
1
Frc. 3. Ir~arnoN oP exzYwunc ecnvrrsr of NNA-PLAZ aY mAbs . NNA-PLAZ wes preincubatod with variable amounts of mAbs, 1E531 (~), SF92 (~) and SF6F10 (/) at 37°C for 2 hr before being assayed . All values are the means of triplicate determinations .
The amino acid sequence of NNA-PLAZ showed a 85%, 77% and 53% sequence homology with that of H. haemachatus DE-I, N. nigricollis CMS-9 and notexin, respectively (ICnaI and IWAxAGA, 1986). Competitive EIA revealed that NNA-PLAZ had an Abs binding affinity 330-fold higher than H. haemachatus DE-I (Fig. 2). This implies that the substituted residues appearing in H. haemachatus DE-I might be related to the epitope(s) of NNA-PLAZ. Further amino acid substitutions occumng in N. nigricollis CMS-9 and noteatin caused them to fail to inhibit the binding of NNA-PLAZ to anti-PLA Z pAbs. Modifications of Tyr-3 and Tyr-63 of NNA-PLAZ (NBS-4) caused some reduction in its binding affinity for pAbs (Fig. 2). Modified derivatives, NBS-2 (modified on Tyr-63), TNP-1 (modified on Lys-6) and TNP-2 (modified on Lys-6 and Lys-65) as well as NBS-4 also exhibited lower Abs binding affinities than the native enzyme (data not shown) . This suggests that Tyr-3, Lys-6, Tyr-63 and Lys-65 might be located on or in proximity to the epitope(s) of NNA-PLAZ. Although the three mAbs (1E531, SF92 and SF6F10) all bound with NNA-PLAZ, the enzymatic activity of NNA-PLAZ was only inhibited by 1E531 and SF92 (Fig. 3). At a molar ratio of l :l, 78% and 90% inhibition for 1E531 and SF92 were observed. However, SF6F10 did not abolish the PLAZ activity. This indicated that the epitope recognized by SF6F10 might be topographically different from that recognized by 1E531 and SF92 . The molar ratios of Ag : Ab of Ag-mAbs complexes which appeared in the incubation mixtures of NNA-PLAZ with two or three kinds of mAbs, calculated from their apparent molecular weights by gel filtration on Sephacryl S-300 column, were 1 :1 (data not shown) . This finding suggests that these mAbs do not bind simultaneously with NNA-PLAZ, and that the epitopes of PLAZ recognized by these three mAbs are located on close or partially overlapping regions. The failure of these three mAbs to react with reduced and S-carboxymethylated NNA-PLAZ indicated that the epitopes recognized by these mAbs are conformational dependent ones . As shown in Table 1, BPB-PLAZ displayed a slight decrease in ai$nities for the three mAbs . Selective modification of tyrosine residues (at positions 3 and 63) and lysine residues (at positions 6 and 65) of NNA-PLAZ all induced a marked reduction in affinities for mAbs . In view of the changes in their lei values, it would seem that lysine residues
156
C. C. YANG et al.
played a more crucial role in the interaction of mAbs with NNA-PLAZ than tyrosine residues . After removal of the N-terminal octapeptide, the Icy value drastically increased. These results suggest that the N-terminal region and the modified residues might belong to the epitope recognized by these mAbs. Alternatively, the inability of PLAZ homologues to compete with NNA-PLAZ for binding mAbs implies that some of the substituted residues in PLAZ homologues are involved in the Ag-mAb interaction. DISCUSSION
In this study, the derivatives of NNA-PLAZ modified either at tyrosine or lysine residues showed a decrease in affinities for anti-PLAZ pAbs and mAbs as compared with native enzyme (Table 1). Removal of the N-terminal octapeptide also affected the epitopes interacting with the mAbs. Our previous finding (YANG and CHANG, 1988) using anti-PLAZ pAbs proved that the N-terminal region is one of epitopes in NNA-PLAZ. These results suggest that the N-terminal region, Tyr-63 and Lys-65 might play a critical role in the Ag-Ab interaction. Although these residues are separated in the peptide sequence, the three-dimensional structure of NNA-PLAZ (WHITE et al., 1990 ; ScoTT et al ., 1990) shows that these residues are brought together by virtue of folding of the polypeptide chain (Fig. 4). Thus, it is likely that these residues themselves and/or their nearby region mutually construct the conformational dependent epitope(s) of NNA-PLAZ. The comparison of the amino acid sequence of NNA-PLAZ to those of PLAZ homologues shows that the greater part of substituted residues occur in the spatial region surrounding the N-terminal region, and residues at homologous positions 63 and 65. The fact that the homologous variants of NNA-PLAZ had a lower reactivity toward anti-PLAZ pAbs and mAbs supports the view that the epitope(s) of NNA-PLAZ might be composed of the N-terminal region, Tyr-63 and Lys-65 and/or their neighbouring regions. Prediction of the probable epitope regions from the hydrophilicity profile (Hoof and WOODS, 1981) TAeLe 1. INHHt1170N oe 'vI-r .na~ .rF NNA-PLAz anvDnva ro mAbs sY CHH~CALLY MODQ+~.D NNA-PLAZ DFRIVATIYES AND PLAz HOMOLOCiUP3
1E531 Native NNA-PLAZ Modified NNA-PLA=: BPB (His-47)' TNP-1 (Lys-6) TNP-2 (Lys-6 & 65) NBS-2 (Tyr-63) NBS-4 (Tyr-3 dc 63) CBIIt PLAZ homologuea : X. haemachatw DE-I N. nigricollis CMS-9 Notexin
(IIM) SF92
IC~p
SF6F10
105
46
45
228 1570 2860 577 741 3409
113 970 2120 386 970 2654
67 1456 2588 427 1966 6062
> 10'
S350 8606 > 10~
6535 5912 > 10`
> 10' > 10'
The numbers in parentheses indicate the positions of modified residues in NNA-PLA=. t NNA-PLAN was cleaved with CNBr, and the N-terminal octapeptide (CBn was separated from the large C-terminal fragment (CBII) (YANO and CFIANG, 1988).
Naja naja atra PhosphoGpase A2
157
Fyc . 4. TERTIARY STRUCTURE OF NNA-PLAZ sxownvc T~ REUrtvE r oceTCOxs of T~ Monu~o
RP3II)UFS. The model is based on the result of Sara et al. (1990) . Numbers in parentheses indicate the positions of Tyr-3, Lys-6, Tyr-63 and Lys-65 in NNA-PLAN.
t â 0 ôT 2
2 -4 v
20
40
60
~
80
v
100
o 120
Sequence position Flc. 5. HYDROPATHY PROtIIL Or NNA-PLA2. The averaged hydropathic indexes were plotted vs the amino acid sequence of NNA-PLAN according to the method of Hour and Woons (1981). The x-axis contains l20 increments, each representing an amino acid in the sequence of NNA-PLA2. The y-axis represents the hydropathic indexes represented as the averages of seven residues . The data points are plotted at the centre of the averaging group from which they were derived. The bar represents the probable epitope regions (floor and Woous, 1981).
158
C . C. YANG er a/.
of the amino sequence of NNA-PLAZ also suggests that segments 1-7 and 62-72 are two of the five candidate epitope regions shown in Fig. 5. The residues of the N-terminal region, Tyr-63 and Lys-65 are conserved residues in NNA-PLA Z and H. haemachatus DE-I (Klxl and Iwwxwcw, 1986); however, competitive RIA revealed that H. haemachatus DE-I has a weak affinity for anti-NNA-PLA Z mAbs . This implies the involvement of other residues in the interaction between mAbs and NNA-PLA Z besides the N-terminal region, Tyr-63 and Lys-65 . In view of the inhibitory capacities of mAbs on PLA Z activity, it is plausible that the other residues of the epitopes recognized by 1E531 and SF92 were neighbouring on or were part of the catalytic site . Consequently, the enzymatic activity of NNA-PLAZ was inhibited. The inability of SF6F10 to inhibit the PLAZ activity suggests that the other residues in its epitope were topographically distant from the catalytic site of NNA-PLAZ . Acknowledgements`This work was supported by the National Science Council, Republic of China . We thank Dr J. Teusoo'r-r' for reading this manuscript, and also Miss F. S . Wu for purification of PLAz enzymes and notexin .
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LraxEa, R. A . (1982) Tapping the immunological repertoire to produce antibodies of predetermined specificity. Nature 299, 592-5% . RsNStseoea, R ., Bxutvn:, S ., Dutcsran, B. W ., DR~vrH, J . and S~ct~x, P. H. (1985) A comparison of the crystal structures of phoapholipase Az from bovine pancreas and Crotales atrox venom . J. biol. Chem . 260, 11,627-11,634. RostareEac, P ., Corroxa+, E., R~univo, B . E ., $GONS, K. R. and Y~rc, C . C. (1983) Dissociation of pharmacological and enzymatic activities of snake venom phospholipases AZ by modification of carboxylate groups . Biochem. Pharmac . 32, 3524-3530 . Scow, D. L., WxiTe, S. P ., OrwiNOwsict, Z ., Yuert, W., GEt .e, M . H . and S~ct ete, P. B . (1990) Interfacial catalysis: the mechanism of phospholipase A2 . Science 250, 1541-1546 . Txuxxts~v, M . M . G . M ., Kwc.tc, K. H ., DxaNrtt, J . and Dutcsrge, B. W . (1990) Structure of an engineered porcine phospholipase AZ with enhanced activity at 2.1 A tnsolution. J. molec . Biol. 216, 42539. Tsnt, I . H ., Wu, S. H. and Lo, T. B . (1981) Complete amino acid sequence of a phospholipase A= from the venom of Naja naja afro (Taiwan cobra) . Toxicon 19, 141-152. W~, M. (1987) Phospholipases, pp . 155-241 . New York : Plenum Press . Wtit~, S . P., Scow, D . L ., t7rwnvowsgt, Z ., Grt,a, M . H . and Stat-©e, P. B . (1990) Crystal structure of cobra venom phospholipase A2 in a complex with transition-state analogue. Science 250, 1560-1563 . YANG, C. C . and Crnwe, L. S . (1984) Tryptophan modification of phosopholipase AZ enzymes and presynaptic neurotoxins from snake venom . J. protein Chem. 3, 195-213 . YeNC, C . C. and CtiANG, L . S. (1988) Role of the N-terminal region in phospholipase Az from Naja naja afro (Taiwan cobra) and Naja nigricollis (spitting cobra) venons . Toxicon 26, 721-731 . YANG, C . C . and C~NC, L. S. (1989) Studies on the status of lysine residues in phospholipase AZ from Naja nçja afro (Taiwan cobra) snake venom . Biochem. J. 262, 855-860. YnNC, C . C . and C~uNC, L. S . (1990) The N-terminal amino group essential for the biological activity of notexin from Notechis scutatus seatatus venom . Biochim . biophys. Acta 1040, 351 . YANG, C. C . and K~NC, K . (1980x) Chemical modification of the histidine residue in basic phospholipase A~ from Naja nigricollis snake venom. Biochim . biophys. Acta 614, 373-388 . YANG, C . C . and K~NC, K . (19806) Chemical modification of the histidine residue in phoapholipase Az from the Hemachatur haemachatus snake venom . Toxicon 18, 529-547. Yertc, C . C ., L1N, M . F . and CtteNC, C . C . (1977) Purification of anti-cobrotoxin antibody by affinity chromatography . Toxicon 15, 512. Y~rrG, C. C ., Ktxc, K . and Sex, T . P. (1981) Chemical modification of lysine and histidine residues in phospholipase AZ from the venom of Naja naja afro (Taiwan cobra) . Toxicon 19, 645-659. YANG, C . C ., KrNC, K., Sex, T. P. and Hseu, W . S . (1982) Lysine modification in snake venom phospholipases A2 . The Snake 14, 110-118 . Yexc, C . C ., HUANG, C . S . and Lam, H . J . (1985) Studies on the status of tyrosyl residues in the phospholipase Az from Naja naja afro and Naja nigricollis snake venons . J. protein Chem . 4, 87-102 .
