Chen-Chung Yang

Dissociation of enzymatic activity from lethality and pharmacological properties by carbamylation of lysines in Naja nigricollis and Naja naja atra snake venom phospholipases A2

Abstract Carbamylation of 9 out of 10 lysine residues in the toxic phospholipase A2 from N. nigricollis venom decreased its lethality at least 8-fold and abolished its direct hemolytic and anticoagulant activities, while the enzymatic activity, as measured on purified substrates, decreased only about 50%. Likewise, carbamylation of 3 out of 5 lysines in the relatively less toxic N. naja atra phospholipase induced detoxification and caused a loss of its blocking activity on the phrenic nerve-diaphragm preparation, while its enzymatic activity on purified substrates was unaltered. Results obtained when 7.4 out of 10 lysines in N. nigricollis phospholipase were carbamylated indicate that basicity is not an absolute requirement for high lethal potency, hemolytic activity or cardiotoxicity. The extent of phospholipid hydrolysis induced in erythrocytes, rabbit plasma, phrenic nerve-diaphragm preparation, brain minces and brain synaptic plasma membranes by incubation with the carbamylated enzymes was in agreement with their enzymatic activities as measured on purified substrates. Levels of phospholipid hydrolysis in heart, lung and kidney of mice given phospholipase intravenously, and in brain synaptic plasma membranes from rats given phospholipase intraventricularly, showed that carbamylated derivatives of N. nigricollis phospholipase A2 lost their ability to reach and/or hydrolyze substrates in vivo. However, the decrease in in vivo phospholipid hydrolysis did not correlate with the decrease in toxicity since, at comparably low levels of phospholipid hydrolysis, some phospholipases were lethal and others were not. Moreover, when intraventricularly administered, both lethal amounts of the native N. naja atra enzyme and its nonlethal carbamylated derivatives produced equally low hydrolysis of synaptic membrane phospholipids. By means of lysine carbamylation, a dissociation between hydrolytic activity and pharmacological properties of phospholipases A2 has been achieved. We suggest, therefore, that the toxicity of pure phospholipases is primarily due to a direct effect which does not correlate with levels of phospholipid hydrolysis and that this direct effect is prominent in the relatively toxic phospholipases while it is less manifest in the relatively non-toxic enzymes.

Effect of modification of one histidine residue on the enzymatic and pharmacological properties of a toxic phospholipase A2 from Naja nigricollis snake venom and less toxic phospholipases A2 from Hemachatus haemachatus and Naja naja atra snake venoms

Modification of one histidine residue at the active site in the phospholipases A2 from N. nigricollis, H. haemachatus and N. naja atra snake venoms reduced the enzymatic activity to 0·–0·8% that of the native species, while the Km values for the first two enzymes listed remained practically unchanged. Also unchanged following modification were the characteristic substrate specificities of the three enzymes. While it appears most likely that the residual activity of the modified enzymes originates from contamination by non-modified species, the modified enzyme might have residual activity of its own. The decrease in enzymatic activity was accompanied by a decrease in all other biochemical and physiological parameters studied such as: direct hemolysis (for N. nigricollis phospholipase), ability to hydrolyze red cell phospholipids, intravenous ld50 in mice, intraventricular ld50 and cd50 in rats, brain phospholipid hydrolysis and ability to block action potentials in eel electroplax. The results support the view that catalytic activity is essential for toxic action.

Chemical modification of lysine and histidine residues in phospholipase A2 from the venom of Naja naja atra (Taiwan cobra)

Abstract The major phospholipase A 2 was isolated from Naja naja atra venom by successive chromatography on SP-Sephadex C-25, DEAE-Sephacel, CM-Sephadex C-25 and SP-Sephadex C-25 columns. The homogeneity was verified by disc electrophoresis and the isoelectric point determined to be 5.2. The specific activity was 3400 U/mg protein, and the ld 50 8 mg/kg mouse. The purified phospholipase A 2 was subjected to lysine modification with cyanate at pH 8.0. After suitable periods (3 and 16 hr), the carbamylated derivatives were separated on a column of DEAE-Sephacel and eight fractions were obtained (DE-1 to DE-8). The results of amino acid analysis showed that one to five Lys-residues were modified. Associated with modification of increasing numbers of Lys-residues were progressive decreases in pI values and marked decreases (3 to > 30-fold) in ld 50 values. However, the decrease in enzymatic activity was slight and antigenic specificity was unaffected. The results show a clear dissociation between enzymatic activity and lethal toxicity. The enzyme was also subjected to chemical modification with p -bromophenacyl bromide. Alkylation of the only His-47 at the active site of the phospholipase A 2 destroys both catalytic activity and lethal toxicity, whereas the antigenicity remained unchanged. Although all the native, Lys-modified and His-modified phospholipases A 2 were perturbed by the presence of Ca 2+ and the difference spectra of Lys-modified DE-6 was similar to that of native phospholipase A 2 , the difference spectra of His-modified enzyme differed greatly from that of the native enzyme. The emission intensity of 8-anilinonaphthalenesulfonate-enzyme complex was altered by increasing concentrations of Ca 2+ , and different results were observed at different pH values of the buffer solution, indicating that Ca 2+ causes pH-dependent conformational changes. The Scatchard plots showed only one kind of specific interaction between 8-anilinonaphthalenesulfonate and native or Lys-modified enzyme (DE-6), and the dissociation constant of Lys-modified DE-6 was similar to that of the native enzyme. On the other hand, the His-modified enzyme lost the ability to bind 8-anilinonaphthalenesulfonate.

Do chemical modifications dissociate between the enzymatic and pharmacological activities of β bungarotoxin and notexin

We have measured enzymatic, hemolytic and anticoagulant activities, lethal potencies and effects on contractions of the phrenic nerve-diaphragm preparation, by chemically modified derivatives of beta bungarotoxin (beta BuTX) and notexin, two presynaptically acting toxins which have PLA2 activity. The following chemical modifications of beta BuTX were tested: alkylation and methylation of histidine 48, alkylation of tryptophan 19, sulfonylation of tyrosine 68, oxidation of methionines 6 and 8, semicarbazide addition under varied conditions to carboxyl groups, varied extents of carbamylation or trinitrophenylation of lysines and guanidination of all lysines with or without trinitrophenylation of the N-terminal asparagine. Only the histidine, tryptophan and tyrosine residues were modified in notexin. The results obtained were compared with those previously obtained using chemically modified derivatives of Naja nigricollis and Naja naja atra PLA2 enzymes which do not have a specific presynaptic site of action. The results with oxidized methionine and lysine-modified derivatives of beta BuTX are supportive of the suggestions of others that the N-terminal region and basic residues away from the enzymatic active region contribute towards the beta type presynaptic neurotoxicity of the PLA2 toxins. Using modified derivatives of beta BuTX and notexin, the dissociations between enzymatic activities and pharmacological properties were not as marked as previously observed with N. nigricollis and N. n. atra PLA2; nevertheless, several dissociations were noted. We conclude that, just as with non-presynaptically acting PLA2 enzymes, some pharmacological actions of presynaptically acting PLA2 toxins may occur independently of phospholipid hydrolysis.

Ethoxyformylation and guanidination of snake venom phospholipases A2: Effects on enzymatic activity, lethality and some pharmacological properties

Lysine residues in the basic and relatively toxic N. nigricollis phospholipase A2 and in the acidic and relatively nontoxic N. n. atra phospholipase A2 were modified by acylation with ethoxyformic anhydride (in the presence or absence of the substrate dihexanoyl lecithin) or guanidination with O-methylisourea. Ethoxyformylation gave rise to some protein fractions in which enzymatic activity was preserved to a greater degree than intraventricular lethality. Guanidination had little effect on the isoelectric point or catalytic activity of either enzyme or on the lethal potency of the N. n. atra enzyme. However, the intraventricular lethality of the N. nigricollis enzyme was decreased much more than was its intravenous lethality, direct hemolytic potency, anticoagulant activity or cardiotoxic action on rat atrium. These results are compared to those previously obtained when the lysines in these two enzymes were carbamylated with potassium cyanate, a procedure which markedly decreased the isoelectric point of the enzymes. It is concluded that charge alone does not account for differences in toxicity. The data also indicate that there are at least two distinct active sites in both enzymes, one being primarily responsible for enzymatic activity and the other(s) associated with lethal and pharmacological effects of the protein. Modification of lysines affects the latter site(s), while having little or no effect on enzymatic activity.

Chemical modification of the histidine residue in phospholipase A2 from the Hemachatus haemachatus snake venom.

Abstract The major phospholipase A2 (DE-I) was isolated from Hemachatus haemachatus (Ringhals) venom and further purified on SP-Sephadex C-25. The homogeneity was verified by disc electrophoresis and the isoelectric point determined to be 7·35. The specific activity was 2950 U mg protein at 30°C and pH8·0. The purified DE-I was subjected to chemical modification with p-bromophenacyl bromide at pH 8·0. Both the enzymatic activity and lethal toxicity were lost; however, the antigenic specificity remained unchanged. Although both ANS and Ca2+ showed pronounced protection on the inactivation process, the mechanism of ANS protection is different from that of Ca2+. Amino acid analysis showed only one out of four His-residues was modified and the modified residue was identified to be His-47 in the sequence. Ultra-violet difference spectra revealed that both native and His-modified DE-I were perturbed by the presence of Ca2+ and the data indicated that a charge effect was responsible for the typical tryptophan blue shift of the Ca2+ bound native DE-I. However, the modified enzyme lost the characteristic tryptophan blue shift suggesting that a titratable group in the vicinity of a tryptophan residue was unable to exert a charge effect. DE-I not only enhanced the emission intensity of ANS dramatically but also shifted the maximum from 480 to 450 nm. On the other hand, the His-modified DE-I did not increase the emission intensity at all. It is suggested tentatively that the hydrophobic pocket in which ANS bound may be the site of the enzyme that interacts with phospholipid.

Effects of modification of tyrosines 3 and 62 (63) on enzymatic and toxicological properties of phospholipases A2 from Naja nigricollis and Naja naja atra snake venoms.

Previously we selectively modified His (48), Arg, Lys, Asp, Glu and Trp residues in the basic phospholipase A2 from Naja nigricollis and the acidic phospholipase A2 from N. n. atra snake venoms. Evidence was obtained for the existence of separate but perhaps overlapping sites responsible, respectively, for their enzymatic and pharmacological properties. We have now modified one or two (Tyr 3, Tyr 62 [63], Tyr 3 + 62 [63]) out of the nine tyrosine residues in these enzymes using p-nitrobenzenesulfonyl fluoride. The derivatives were separated by HPLC, and modified residues determined by amino acid analysis. Enzymatic activity was tested on lecithin--Triton mixed micelles, egg yolk and heart and diaphragm homogenates. The N. nigricollis modified derivatives retained a greater percentage of their enzymatic activities than did the N. n. atra derivatives and also a greater percentage of their activity on natural substrates than on lecithin--Triton mixed micelles. The greatest loss in activity resulted when both tyrosines were modified and the least when tyrosine 3 was modified. Modification of tyrosine 62 of N. nigricollis caused a much greater loss of intraventricular lethal potency than of enzymatic activity, whereas modification of tyrosine 3 of N. n. atra increased lethal potency over six-fold while enzymatic activity decreased about 60%. Examples of dissociation between enzymatic and pharmacological potencies were also noted when hemolytic, anticoagulant and cardiotoxicity on isolated ventricular muscle were measured. The extents of phospholipid hydrolysis were relatively low in brain homogenates, synaptic plasma membranes and heart ventricular muscle. However, they were similar for the native enzymes and all of the tyrosine modified derivatives. These tyrosines do not appear to be part of the enzymatic active site, even though they are thought to be associated with substrate and calcium binding. These results strengthen our earlier conclusion that some pharmacological effects of phospholipase A2 are not due to enzymatic hydrolysis, and that there are separate but perhaps partly overlapping sites for enzymatic and pharmacological activities.

Role of the N-terminal region in phospholipases A2 from Naja naja atra (Taiwan cobra) and Naja nigricollis (spitting cobra) venoms

The N-terminal alpha-amino groups of two phospholipases A2 (PLA2) from Naja naja atra and Naja nigricollis venoms were selectively modified with trinitrobenzene sulfonic acid, and the modified derivatives were separated by high performance liquid chromatography (HPLC). Trinitrophenylated (TNP) derivatives contained only one TNP group in the alpha-amino group of Asn-1 and showed a marked decrease in enzymatic activity. PLA2 enzymes were cleaved with CNBr, and the N-terminal octapeptide was separated from the large C-terminal fragment by HPLC. Removal of the N-terminal octapeptide from PLA2 enzymes caused a precipitous decrease in enzymatic activity. Enzyme immunoassay and double immunodiffusion revealed that the N-terminal octapeptide is one of the antigenic determinants of PLA2 enzymes. The presence of dihexanoyllecithin influenced the interaction between PLA2 enzymes and 8-anilinonaphthalene sulfonate (ANS), indicating that ANS-binding site of PLA2 enzymes is at or near the substrate binding site. Modification of the N-terminal region perturbed the substrate binding and the binding ability for ANS. The modified derivatives retained their affinity for Ca2+, indicating that the N-terminal region is not involved in Ca2+-binding. A fluorescence study revealed that the alpha-amino group is near Trp residue(s) and that the N-terminal region is important for stabilizing the architectural environment of the Trp residue(s). The results, together with the proposal that Trp residues in PLA2 enzymes are involved in substrate binding, suggest that the N-terminal region of PLA2 enzymes is involved in substrate binding and in maintaining a functional active site.

Effect of carboxylate group modification on enzymatic and cardiotoxic properties of snake venom phospholipases A2

By treating Naja nigricollis and Naja naja atra phospholipase A2 with carbodiimide and semicarbazide, we obtained derivatives having varied numbers of modified carboxylate groups. When tested on artificial and natural substrates, derivatives of both enzymes with a modified carboxylate group at the active site (Asp-49) retained little enzymatic activity (1/41 to 10%). However, the derivatives of N. nigricollis also lost most of their lethal potency (5% of native), while those of N. n. atra retained considerable lethality (29%). Carboxyl modification with protection of Asp-49 in N. n. atra enzyme resulted in a derivative with lethal potency equal to or greater than the native enzyme and enzymatic activity which was low on all substrates (12-17% of native). Similar protection of Asp-49 at the active site in N. nigricollis enzyme produced a derivative with decreased enzymatic activity on artificial substrate (22% of native) and decreased lethality (17-33% of native), but with full enzymatic activity on natural substrates. When tested on electrical and mechanical properties of the isolated perfused heart and the isolated ventricle muscle wall, the derivatives of both enzymes retained considerably more of the cardiotoxic activity than would have been expected based on their residual enzymatic activity. The one exception occurred with the least modified N. nigricollis derivative which had an unaltered Asp-49, this enzyme retained both cardiotoxic activity and full enzymatic activity on natural substrates. The extent of phospholipid hydrolysis following treatment was measured in the isolated heart preparation and in hearts removed from mice following i.v. injection of the phospholipases. Very low levels of phospholipid hydrolysis were observed and no correlation could be made between the extent of hydrolysis and the pharmacological potencies of these enzymes. Modification of the enzymatic active site, whether of Asp-49 in this study of His-48 in prior studies, leads to a large decrease in both enzymatic activity and lethal potency. Asp and Glu residues outside of the enzymatic site contribute significantly to the lethal potency of the N. nigricollis enzyme and to the enzymatic activity of the N. n. atra enzyme. Based on these and previous data we conclude that changes in isoelectric points are not responsible for altered lethal potencies following chemical modification and that some pharmacological effects of snake venom phospholipases A2 are due to a non-enzymatic action, suggesting two distinct but perhaps overlapping active sites.

Venom constituents of Notechis scutatus scutatus (Australian tiger snake) from differing geographic regions

Column chromatography and polyacrylamide gel electrophoresis of Notechis scutatus scutatus venom showed that the venoms from different geographical locations had variations in their constituents. The venom collected from South Australia region contained both notexin and notechis II-5. The relative quantity of notechis II-5 was about three times that of notexin. On the other hand, the venom from Victoria region contained large amounts of notexin, but lacked notechis II-5. Instead, an unknown nontoxic protein, designated as notechis II-5b, exhibiting weak phospholipase A2 activity appeared in the position of notechis II-5 elution. This protein had an N-terminal sequence of N-L-I-Q-L-S-N-M-I-K-C-A-I-P-G-S-Q-P-L-F, sharing 45% homology with notexin and notechis II-5 and 60% homology with notechis II-1. The antibodies raised against Trp-modified notexin inhibited the enzymatic activities of notexin and notechis II-5 by 88 and 68%, respectively. However, the affinity of notexin for the antibodies was nine-fold greater than that of notechis II-5. This result is contrary to the previous finding (Mollier et al., FEBS Lett. 250, 479-482, 1989) in which notexin and notechis II-5 had similar binding affinities for antibodies raised against native notexin. This observation suggests that the antibodies prepared in this study could differentiate between isoforms of notexin.