Snake Venom PLA2 Research Articles

Inflammatory effects of snake venom myotoxic phospholipases A 2

Snake venom phospholipases A 2 (PLA 2) show a remarkable functional diversity. Among their toxic activities, some display the ability to cause rapid necrosis of skeletal muscle fibers, thus being myotoxic PLA 2s. Besides myotoxicity, these enzymes evoke conspicuous inflammatory and nociceptive events in experimental models. Local inflammation and pain are important characteristics of snakebite envenomations inflicted by viperid and crotalid species, whose venoms are rich sources of myotoxic PLA 2s. Since the discovery that mammalian PLA 2 is a key enzyme in the release of arachidonic acid, the substrate for the synthesis of several lipid inflammatory mediators, much interest has been focused on this enzyme in the context of inflammation. The mechanisms involved in the proinflammatory action of secretory PLA 2s are being actively investigated, and part of the knowledge on secretory PLA 2 effects has been gained by using snake venom PLA 2s as tools, due to their high structural homology with human secretory PLA 2s. The inflammatory events evoked by PLA 2s are primarily associated with enzymatic activity and to the release of arachidonic acid metabolites. However, catalytically inactive Lys49 PLA 2s trigger inflammatory and nociceptive responses comparable to those of their catalytically active counterparts, thereby evidencing that these proteins promote inflammation and pain by mechanisms not related to phospholipid hydrolysis nor to mobilization of arachidonic acid. These studies have provided a boost to the research in this field and various approaches have been used to identify the amino acid residues and the specific sites of interaction of myotoxic PLA 2s with cell membranes potentially involved in the PLA 2-induced inflammatory and nociceptive effects. This work reviews the proinflammatory and nociceptive effects evoked by myotoxic PLA 2s and their mechanisms of action.

Structural Effects of Covalent Inhibition of Phospholipase A 2 Suggest Allosteric Coupling between Membrane Binding and Catalytic Sites

Phospholipase A 2 (PLA 2) binds to membranes and catalyzes phospholipid hydrolysis, thus initiating the biosynthesis of lipid-derived mediators of inflammation. A snake-venom PLA 2 was completely inhibited by covalent modification of the catalytic histidine 48 by p-bromophenacyl bromide. Moreover, His 48 modification affected PLA 2 structure, its membrane-binding affinity, and the effects of PLA 2 on the membrane structure. The native PLA 2 increased the order parameter of fluid membranes, whereas the opposite effect was observed for gel-state membranes. The data suggest membrane dehydration by PLA 2 and the formation of PLA 2-membrane hydrogen bonding. The inhibited PLA 2 had lower membrane-binding affinity and exerted weaker effects on membrane hydration and on the lipid-order parameter. Although membrane binding resulted in formation of more flexible α-helices in the native PLA 2, which corresponds to faster amide hydrogen exchange, the modified enzyme was more resistant to hydrogen exchange and experienced little structural change upon membrane binding. The data suggest that 1), modification of a catalytic residue of PLA 2 induces conformational changes that propagate to the membrane-binding surface through an allosteric mechanism; 2), the native PLA 2 acquires more dynamic properties during interfacial activation via membrane binding; and 3), the global conformation of the inhibited PLA 2, including the α-helices, is less stable and is not influenced by membrane binding. These findings provide further evidence for an allosteric coupling between the membrane-binding (regulatory) site and the catalytic center of PLA 2, which contributes to the interfacial activation of the enzyme.

Characterization and cloning of a novel phospholipase A 2 from the venom of Trimeresurus jerdonii snake

A phospholipase A 2 (PLA 2), called jerdoxin, was isolated from Trimeresurus jerdonni snake venom and partially characterized. The protein was purified by three chromatographic steps. SDS–polyacrylamide gel electrophoresis in the presence or absence of dithiothreitol showed that it had a molecular mass of 15 kDa. Jerdoxin had an enzymatic activity of 39.4 μmol/min/mg towards egg yolk phosphatidyl choline (PC). It induced edema in the footpads of mice. In addition, jerdoxin exhibited indirect hemolytic activity. About 97% hemolysis was observed when 2 μg/ml enzyme was incubated for 90 min in the presence of PC and Ca 2+. No detectable hemolysis was noticed when PC was not added. Ca 2+ was necessary for jerdoxin to exert its hemolytic activity, since only 52% hemolysis was seen when Ca 2+ was absent in the reaction mixture. Furthermore, jerdoxin inhibited ADP induced rabbit platelet aggregation and the inhibition was dose dependent with an IC 50 of 1.0 μM. The complete amino acid sequence of jerdoxin deduced from cDNA sequence shared high homology with other snake venom PLA 2s, especially the D49 PLA 2s. Also, the residues concerned to Ca 2+ binding were conserved. This is the first report of cDNA sequence of T. jerdonii venom PLA 2.

Injury and recovery of fast and slow skeletal muscle fibers affected by ACL myotoxin isolated from Agkistrodon contortrix laticinctus (Broad-Banded Copperhead) venom

The response of different types of skeletal muscle fibers to a snake venom PLA 2 myotoxin was tested in vivo by injecting ACL myotoxin (ACLMT) into mice. Both the soleus (slow-twitch) and gastrocnemius (fast-twitch) were examined at different time periods (3 h, 3 and 21 d) after the injection. All animals received 5 mg/kg myotoxin into the subcutaneous lateral region of the right hind limb, near the Achilles tendon; contralateral muscles were used as controls. Cross-sections (10 μm) of frozen muscle tissue were cut from the medial region of the muscle. Alternate serial sections were stained either with toluidine blue or for acid phosphatase, myofibrillar ATPase activity after alkali (pH 10.3) or acid preincubation (pH 4.3), succinate dehydrogenase or acetylcholinesterase. Several stages of necrosis were observed 3 h after ACLMT injection, in both superficial and deep regions of both muscles. In these same regions 3 d after injection, clusters of regenerated muscle fibers were present, and some of them presented AChE activity. Twenty-one days after ACLMT injection the muscle fibers of soleus and gastrocnemius presented only chronic signs of damage such as split fibers and centralized nuclei. Using m-ATPase reactions it was possible to determine that both muscle fiber types I and II were injured in both muscles. The number of type IIC fibers was significantly increased, and the number of type II fibers significantly decreased in the gastrocnemius 21 d after ACLMT injection, suggesting a change in muscle fiber type from type II to type I, through type IIC. The increased number of type IIC fibers and the presence of AChE activity in clusters of regenerating fibers and split fibers indicate that injury by ACLMT produces axonal remodeling and muscle fiber type change.

INJURY AND RECOVERY OF FAST AND SLOW MUSCLE FIBERS AFFECTED BY A SNAKE VENOM MYOTOXIN 375

To study the response of individual muscle fiber types to snake venom PLA2 myotoxins, we have tested the effect of ACLMT (ACL myotoxin fromAgkistrodon contortrix laticinctus) on soleus andgastrocnemius mice muscles. All animals received 5 mg of ACLMT/kg into the subcutaneous lateral region of the right hind limb, near the Achilles tendon. The contralateral muscles were used as control. Frozen muscles were cut at medial region in Cryostat and alternate serial cross-sections stained with Toluidine Blue and for Acid Phosphatase, Myofibrillar ATPase activity, Succinate Dehydrogenase and Acetilcholinesterase. Signs of fiber injury were identified, three hours after ACLMT injection, in both muscles. Three days after, these muscles showed clusters of recovered muscle fibers. Twenty-one days after soleus and gastrocnemius presented only chronic signs of damage as split fibers and centralized nucleus. By m-ATPase reactions was possible to identify that both muscle fiber types I and II were injured in both muscles by ACLMT. Significative increased number of fiber type IIC (17.37 ± 4.11versus 1.0 ± 0.6; p=0.008, paired Student t-test) and decrease of type II (60.7 ± 8.8 versus 76.8 ± 10.1; p=0.01, paired Student t-test), suggest muscle fiber type change from type II to type I through type IIC. Although ACLMT is known by its myotoxic activity, the results presented here showed that it can also be used as model to induce axonal remodeling and muscle fiber type change in both fast and slow muscle fibers of mice.

Accelerated evolution in the protein-coding regions is universal in crotalinae snake venom gland phospholipase A2 isozyme genes.

The nucleotide sequences of four genes encoding Trimeresurus gramineus (green habu snake, crotalinae) venom gland phospholipase A2 (PLA2; phosphatidylcholine 2-acylhydrolase, EC 3.1.1.4) isozymes were compared internally and externally with those of six genes encoding Trimeresurus flavoviridis (habu snake, crotalinae) venom gland PLA2 isozymes. The numbers of nucleotide substitutions per site (KN) for the noncoding regions including introns were one-third to one-eighth of the numbers of nucleotide substitutions per synonymous site (KS) for the protein-coding regions of exons, indicating that the noncoding regions are much more conserved than the protein-coding regions. The KN values for the introns were found to be nearly equivalent to those of introns of T. gramineus and T. flavoviridis TATA box-binding protein genes, which are assumed to be a general (nonvenomous) gene. Thus, it is evident that the introns of venom gland PLA2 isozyme genes have evolved at a similar rate to those of nonvenomous genes. The numbers of nucleotide substitutions per nonsynonymous site (KA) were close to or larger than the KS values for the protein-coding regions in venom gland PLA2 isozyme genes. All of the data combined reveal that Darwinian-type accelerated evolution has universally occurred only in the protein-coding regions of crotalinae snake venom PLA2 isozyme genes.

Conodipine-M, a Novel Phospholipase A2 Isolated from the Venom of the Marine Snail Conus magus

We describe the purification and first biochemical characterization of an enzymatic activity in venom from the marine snail Conus magus. This enzyme, named conodipine-M, is a novel phospholipase A2 with a molecular mass of 13.6 kDa and is comprised of two polypeptide chains linked by one or more disulfide bonds. The amino acid sequence of conodipine-M shows little if any homology to other previously sequenced phospholipase A2 enzymes (PLA2s). Conodipine-M thus represents a new group of PLA2s. This is remarkable, since conodipine-M displays a number of properties that are similar to those of previously characterized 14-kDa PLA2s. The enzyme shows little, if any, phospholipase A1, diacyglycerol lipase, triacylglycerol lipase, or lysophospholipase activities. Conodipine-M hydrolyzes the sn-2 ester of various preparations of phospholipid only in the presence of calcium and with specific activities that are comparable to those of well known 14-kDa snake venom and pancreatic PLA2s. The Conus enzyme binds tightly to vesicles of the negatively charged phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphomethanol and catalyzes the hydrolysis of this substrate in a processive fashion. Conodipine-M does not significantly discriminate against phospholipids with unsaturated versus saturated fatty acids at the sn-2 position or with different polar head groups. Linoleoyl amide and a phospholipid analog containing an alkylphosphono group at the sn-2 position are potent inhibitors of conodipine-M. We suggest that the functional resemblance of conodipine-M to other PLA2s might be explained by the utilization of similar catalytic residues.

Cloning and expression of a membrane receptor for secretory phospholipases A2.

Snake venom and mammalian secretory phospholipases A2 are structurally related enzymes that have been associated with several toxic (neurotoxicity, myotoxicity, etc.), pathological (inflammation, hypersensitivity, etc.), or physiological (contraction, proliferation, etc.) processes. We have previously shown that snake venom PLA2s have specific high affinity receptors. Here, we report the molecular cloning of one of these PLA2 receptors (molecular mass approximately 180 kDa), previously purified from rabbit skeletal muscle. It is a membrane protein with a N-terminal cysteine-rich domain, a fibronectin type II domain, eight repeats of a carbohydrate recognition domain, a unique transmembrane domain, and a intracellular C-terminal domain. The 1458-residue PLA2 receptor, expressed in transfected cells, binds svPLA2 with very high affinities (Kd values approximately 10-20 pM). It also tightly binds the two structural types of msPLA2s, i.e. pancreatic PLA2 and synovial PLA2 (Kd approximately 1-10 nM). This receptor might have a key role in normal and pathological actions of secretory PLA2s.

Darwinian evolution of Trimeresurus flavoviridis venom gland phospholipase A2 isozymes

Abstract

Toxic effects of human pancreatic and snake and bee venom phospholipases A 2 on MCF-7 cells in culture

The toxicity of phospholipase A 2 (PLA 2) has been suggested to be involved in the pathology of a number of severe diseases including septic shock and acute pancreatitis. However, testing the toxicity of these substances is difficult in vivo. In the present study we compared the toxicity of PLA 2s from three snake venoms, bee venom and human pancreas on MCF-7 cells grown in culture. Tetrazolium microculture assays were developed to test the cytostatic and cytotoxic effects of PLA 2 on MCF-7 cells. These tests are based on the ability of viable cells to reduce a tetrazolium-based compound MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] to a blue formazan product. Leakage of lactate dehydrogenase (LD) from the cells into the culture medium was also measured. There were marked differences in the toxicity of the PLA 2s tested. Cobra ( Naja mosambique mosambique) venom PLA 2 was toxic to the cells at a concentration of 4.5 U/ml. Light microscopic changes were seen in the injured cells after 3 hr treatment. Sixty-seven per cent of cells were dead after 24hr treatment. Treatment for 4 hr caused irreversible changes in the cells. Leakage of LD was noted from 4 hr onwards. Other snake ( Crotalus adamanteus and Laticauda semifasciata) venom PLA 2s, even after continuous exposure to 4.5 U/ml caused only slight decreases in values obtained in the MTT test. No morphologic changes suggesting a cytotoxic effect were seen. PLA 2 from bee ( Apis mellifera) venom had no toxic effect, either. Continuous exposure of cells to human pancreatic PLA 2 caused a 15% decrease in the MTT-test. The treated cells appeared morphologically normal and after discontinuation of the treatment their growth rate was equal to the control cells.

Effect of aristolochic acid on arachidonic acid cascade and in vivo models of inflammation

Recently, it has been reported that aristolochic acid inhibits phospholipase A2 (PLA2in vitro and also decrease either oedema induced by snake venom and human synovial fluid PLA2. The aim of this research was to study the antiiflammatory activity of aristolochic acid and to investigate the effect of the alkaloid on the enzymes involved in the release of eicosanoids. Our results demonstrate that aristolochic acid is able to inhibit inflammation induced by immunological, immune complexes, and nonimmunological agents such as carrageenan or croton oil. We suggest that one mechanism of the antiinflammatory activity of alkaloid may be directly blocking PLA2 catalyzed release of arachidonic acid. Moreover, this study demonstrates that aristolochic acid could also inhibit other steps involved in eicosanoids release such as cyclooxygenase and lipooxygenase pathways.

Human recombinant non pancreatic secreted platelet phospholipase A2 has anticoagulant activity in vitro on human plasma

Extracellular phospholipase A2 is secreted from the platelets upon activation by a stimulus such as thrombin. The secreted enzyme has been recently cloned and the recombinant protein produced. Snake venom PLA2 effect on platelet and coagulation has been extensively studied (for review see 3,4) and it has been proposed that the anticoagulant phospholipases may inhibit coagulation by competing with clotting proteins for the lipid surface. Structure function relationship for PLA2s with anticoagulant activity indicates that the activity is conferred by positively charged aminoacids between residues 54 and 77. The corresponding segment of human recombinant secreted platelet PLA2 (r-hnps-PLA2) possesses five positively charged aminoacids in this region being at the identical positions to those of PLA2s with known anticoagulant activities. Here using human and rat plasma we have demonstrated for the first time that the recombinant human extracellular secreted platelet PLA2 increases activated partial thromboplastin time but not prothrombin time.

Recombinant human type II phospholipase A 2 lacks edema producing activity in rat

The rat paw edema-inducing, acute inflammatory activity of four snake venom phospholipase A 2s (PLA 2) ( Naja naja, Naja mocambique mocambique, Crotalus atrox and recombinant Naja naja naja) and of recombinant human type II PLA 2 (rh-PLA 2) found in rheumatoid synovial fluid, were compared after a bolus subplantar injection. The snake venom-derived PLA 2s, including the recombinant Naja naja naja, were potent inducers of paw edema. On the other hand, when given in similar amounts (protein and/or enzymatic activity), the rh-PLA 2 did not produce paw edema. Furthermore, the addition of Naja naja PLA 2, blood plasma from rats with adjuvant arthritis or the E. coli-based enzymatic incubation mixture used to measure PLA 2 activity to the injection mixture containing rh-PLA 2, did not result in paw edema-inducing activity. These results suggest that the lack of paw edema-inducing activity of rh-PLA 2, may be due to significant structural differences between snake venom PLA 2s and human PLA 2 which allow snake venom PLA 2s, but not the human II PLA 2, to express inflammatory activity as measured by paw edema in rat induced by a bolus injection.

Antiflammins: Anti-inflammatory activity and effect on human phospholipase A 2

Two anti-inflammatory peptides (antiflammins) corresponding to a high amino acid similarity region between lipocortin I and uteroglobin were tested for their ability to inhibit purified human synovial fluid phospholipase A 2 (HSF-PLA 2). No inhibitory activity was observed, even at such high concentrations of peptides as 50 μM. When antiflammins were preincubated with the enzyme and/or the substrate, no HSF-PLA 2 inhibition was detected. In vivo anti-inflammatory activity of these peptides was evaluated in several experimental models of inflammation induced by carrageenan, croton-oil, oxazolone and Naja naja naja venom phospholipase A 2 (PLA 2). In contrast to the in vitro results, anti-inflammatory activity was observed in all tests, except when inflammation was induced by snake venom PLA 2. Taken together, our results do not support the hypothesis that the in vivo anti-inflammatory effect of antiflammins is directly related to inhibition of PLA 2 activity.

Phospholipase A 2 inhibition by alkylbenzoylacrylic acids

3-(4-Alkylbenzoyl)acrylic acids (ABAAs) were synthesized by acylation of alkylbenzenes with maleic anhydride and then screened in vitro for inhibition of phospholipase A 2 (PLA 2) from snake venom and from porcine pancreas. The inhibitory potency of ABAAs increased with the length of the alkyl residues resulting in IC 50 values of between 10 −7 and 10 −4 mol/L. The most potent inhibitors of the snake venom PLA 2 were the 4-( n)-hexadecyl and octadecyl (OBAA) derivatives. Kinetic experiments referred to a time-dependent inhibitory reaction. Irreversibility was examined by dilution and dialysis. A molar ratio of inactivation of OBAA of nearly 20 was estimated. Double reciprocal replots of the apparent inactivation constants to the concentration of OBAA gave a (pseudo) first order rate constant of inactivation of 2.3 min −1. For the dissociation constant of the enzyme-inhibitor intermediate, a value of 6 × 10 −6mol/L was obtained. On the other hand, the PLA 2 from porcine pancreas seemed hardly to be inhibited by ABAAs. The present data are discussed in relation to the proposed model for PLA 2 inactivation by manoalide. In human PMNs leukotriene B 4 and 5-HETE production was essentially reduced. In human platelets the thrombin-induced TxA 2 production was reduced. Since these effects disappeared after addition of arachidonic acid, these findings refer to a PLA 2 inhibition. The immunologically induced bronchospasm in guinea pigs was significantly and dose-dependently inhibited by OBAA. This indicates that ABAAs might be useful in treating allergic diseases, such as asthma, eczema, allergic shock and others.

A prediction of the tertiary structure of human phospholipase A 2 from synovial fluid and a model of substrate binding

The tertiary structure of human Phospholipase A 2 (PLA 2) from synovial fluid has been predicted by modelling the sequence onto the structure of the homologous snake venom PLA 2 structure ( Crotalus atrox). A model of substrate binding to the protein has been on the predicted structure of the apoenzyme. These modelled structures are discussed.

Characterization of a high affinity binding site for pancreatic-type phospholipase A2 in the rat. Its cellular and tissue distribution.

We showed in an earlier study (Arita, H., Hanasaki, K., Nakano, T., Oka, S., Teraoka, H., and Matsumoto, K. (1991) J. Biol. Chem. 266, 19139-19141) that there is a high affinity and specific binding site for mammalian group I phospholipase A2 (PLA2-I) in Swiss 3T3 fibroblast cells. Analysis of the cellular distribution in rat using 125I-PLA2-I as a radioligand indicated the presence of this site in various cells, including vascular smooth muscle cells (VSMC), vascular endothelial cells, synovial cells, chondrocytes, and gastric mucosal cells. Scatchard analysis of rat VSMC revealed the existence of a single class of binding site with a Kd value of 1.60 nM and a Bmax value of 51.0 fmol/10(6) cells. The mammalian mature type of PLA2s-I derived from several animal species specifically recognized the same site in cells and stimulated DNA synthesis, whereas its inactive zymogen, mammalian group II PLA2s, and snake and bee venom PLA2s showed much lesser activities. 125I-PLA2-I bound to VSMC was rapidly internalized and subsequently released from the cells as trichloroacetic acid-soluble radioactivity. Down-regulation of the PLA2-I site was observed in the treatment of VSMC with cAMP-elevating agents, as well as glucocorticoids. Affinity labeling experiments indicated that PLA2-I binds to a single polypeptide with a mass of approximately 200,000 daltons. These results suggest a novel PLA2-I action on the cellular function via its specific binding site.

Pharmacologic modulation of D-49 phospholipase A2-induced paw edema in the mouse.

Paw edema was produced in CD-1 mice by the injection of 0.3 micrograms of snake venom PLA2 (A.p. piscivorus D-49) into the hind paw. Edema peaked at 10 min, remained elevated until 60 min, and then declined slowly. The PLA2 inhibitors, luffariellolide and aristolochic acid, reduced the edema but only when coinjected with the PLA2. The histamine/serotonin antagonists were the most effective drug class against PLA2-induced paw edema. The PAF antagonists, CV-6202 (iv) and kadsurenone (coinjected) reduced the PLA2-induced edema, whereas high doses of the corticosteroids, dexamethasone and hydrocortisone, were also effective. NSAIDs only partially inhibited the paw edema. The LO/CO inhibitors yielded varying activities, with only BW755C and NDGA inhibiting the edema. These results suggest that PLA2 induces paw edema in the mouse via the action of several classes of inflammatory mediators.

Comparison of enzymatic and pharmacological activities of lysine-49 and aspartate-49 phospholipases A 2 from Agkistrodon piscivorus piscivorus snake venom

The basic Lys-49 phospholipase A 2 (PLA 2) from Agkistrodon pisciuorus piscivorus venom is homologous to the basic Asp-49 PLA 2 from the same venom as well as other snake venom PLA 2 enzymes. It differs, however, in several respects, most important being replacement of the previously invariant Asp-49 at the calcium binding site by Lys, resulting in a reversed order of addition of calcium and phospholipid, phospholipid binding first. Although the preferences for phospholipid substrates of the two enzymes are identical, the apparent V max of the Lys-49 PLA 2 was only 1.4 to 3% that of the Asp-49 enzyme. Similarly, the Lys-49 PLA 2, compared to the Asp-49 PLA 2 had < 3% of the intraventricular lethal potency and 4% of the anticoagulant activity. The intravenous lethal potency of the Lys-49 enzyme was 20% that of the Asp-49 PLA 2 and both had little direct hemolytic activity. In contrast, both enzymes were approximately equipotent on the phrenic nerve-diaphragm preparation and on the isolated ventricle strip of the heart. On the cardiac and neuromuscular preparations, the effects of the Asp-49 PLA 2 were accompanied by hydrolysis of phosphatidylcholine and phosphatidylethanolamine, whereas no phospholipid hydrolysis was observed with the Lys-49 PLA 2. Evaluation of the present results, along with earlier findings using Asp-49 PLA 2 enzymes from Naja nigricollis, Hemachatus haemachatus and Naja naja atra venoms, allows us to conclude that: (1) The A. p. piscivorus Asp-49 PLA 2 enzyme resembles the Asp-49 enzymes fron N. n. atra and H. haemachatus. In contrast, the A. p. piscivorus Lys-49 PLA 2 has much lower enzymatic and anticoagulant activities than the Asp-49 enzymes, but equal cardiotoxic and junctional effects. (2) In contrast to some previous suggestions, basic PLA 2 enzymes are not necessarily more toxic than neutral or acidic enzymes. (3) Pharmacological effects upon the heart and phrenic nerve-diaphragm preparation correlate neither with in vitro measurements of PLA 2 activity nor with actual levels of phospholipid hydrolysis in the heart or diaphragm. This suggests that PLA 2 enzymes exert effects independent of phospholipid hydrolysis.

Dissociation of pharmacological and enzymatic activities of snake venom phospholipases A 2 by modification of carboxylate groups

The carboxylate groups in an acidic and in a basic phospholipase A 2 (PLA 2) enzyme, purified, respectively, from Naja naja atra and Naja nigricollis snake venoms, were modified with carbodiimide and semicarbazide. The derivatives modified at pH 3.5 and pH 5.5 had less than 1% ( N. nigricollis) or 2% ( N. n. atra) residual enzymatic activity, whereas 12–16% enzymatic activity remained following modification at pH 5.5 in the presence of Ca 2+. In marked contrast, these derivatives retained variable, but significantly greater, levels of lethal potency, hemolytic and anticoagulant activities, and abilities to block indirectly and directly induced contractions of the diaphragm. By this modification of aspartic and glutamic acid residues we have, for the first time, obtained derivatives of PLA 2 which selectively retain greater pharmacological activity relative to enzymatic activity. Previously, we had found that modification of lysine and arginine residues produced derivatives which retain enzymatic activity but show a loss of pharmacological properties. These finclings suggest that some pharmacological effects of snake venom PLA 2 enzymes are due to a non-enzymatic action, suggesting two distinct but perhaps overlapping active sites.