Dissociation of enzymatic activity from toxic properties of the most basic phospholipase A2 from Vipera russelli snake venom by guanidination of lysine residues

Phospholipase A2 (PLA2) from Naja naja atra (Taiwan cobra) snake venom was subjected to lysine modification with trinitrobenzene sulphonic acid (TNBS), and two major trinitrophenylated (TNP) derivatives, TNP-1 and TNP-2, were separated by h.p.l.c. TNP-1 contained only one TNP group on Lys-6 and showed a marked decrease in enzymic activity, but still retained 45% of the lethal toxicity. Both Lys-6 and Lys-65 were modified in TNP-2, and modification of Lys-65 caused a further reduction of the lethal toxicity to 12.6%. However, the antigenicity of both TNP-1 and TNP-2 remained unchanged. The reactivity of Lys-6 and Lys-65 toward TNBS was greatly enhanced by Ca2+ and dihexanoyl-lecithin, suggesting that the two Lys residues are not directly involved in the binding of Ca2+ and substrate. The modified derivatives retained their affinity for Ca2+, indicating that Lys-6 and Lys-65 did not participate in the Ca2+ binding. The TNP derivatives could be regenerated with hydrazine hydrochloride. The biological activities of the regenerated PLA2 are almost the same as those of native PLA2. These results indicate that Lys-6 and Lys-65 are important for the biological activities of PLA2, and incorporation of a bulky TNP group on Lys-6 and Lys-65 might give rise to a distortion of the active conformation of PLA2.

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Functional involvement of Lys-6 in the enzymatic activity of phospholipase A2 fromBungarus multicinctus (Taiwan banded krait) snake venom

Journal of Protein Chemistry ◽

10.1007/bf01890463 ◽

1994 ◽

Vol 13 (7) ◽

pp. 641-648 ◽

Cited By ~ 7

Author(s):

Long-sen Chang ◽

Kou-wha Kuo ◽

Shinne-ren Lin ◽

Chun-chang Chang

Keyword(s):

Enzymatic Activity ◽

Phospholipase A2 ◽

Snake Venom ◽

Functional Involvement

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Comparison of a relatively toxic phospholipase A2 from Naja nigricollis snake venom with that of a relatively non-toxic phospholipase A2 from Hemachatus haemachatus snake venom—II. Pharmacological properties in relationship to enzymatic activity

Toxicon ◽

10.1016/0041-0101(81)90055-6 ◽

1981 ◽

Vol 19 (3) ◽

pp. 439

Author(s):

G. Habermehl

Keyword(s):

Enzymatic Activity ◽

Phospholipase A2 ◽

Snake Venom ◽

Pharmacological Properties

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Enzymatic Activity And Brine Shrimp Lethality Of Venom From The Large Brown Spitting Cobra (Naja Ashei) And Its Neutralization By Antivenom

10.21203/rs.3.rs-20006/v1 ◽

2020 ◽

Author(s):

Mitchel Otieno Okumu ◽

James Mucunu Mbaria ◽

Joseph Kangangi Gikunju ◽

Paul Gichohi Mbuthia ◽

Vincent Odongo Madadi ◽

...

Keyword(s):

Enzymatic Activity ◽

Phospholipase A2 ◽

Snake Venom ◽

Brine Shrimp ◽

Dendroaspis Angusticeps ◽

Brine Shrimp Lethality ◽

Brine Shrimp Lethality Assay ◽

Comparison Results ◽

Snake Venom Phospholipase ◽

Naja Haje

Abstract Objective There has been little focus on the enzymatic and lethal activities of Naja ashei venom and their neutralization by antivenom. This study aimed to determine the snake venom phospholipase A2/svPLA2 activity and brine shrimp lethality of N. ashei venom and their neutralization by two antivenoms (I and II). The venom of other snakes in East Africa including the puff adder (Bitis arietans), green mamba (Dendroaspis angusticeps), black mamba (Dendroaspis polylepis), Egyptian cobra (Naja haje), red spitting cobra (Naja pallida), and the Eastern forest cobra (Naja subfulva) were used for comparison. Results: N. subfulva venom had the highest svPLA2 activity while D. angusticeps venom had the least activity. N. subfulva venom was the most toxic in the 24-hour brine shrimp lethality assay (BSLA), while N. ashei venom was the most toxic in the 48 and 72-hour assays. N. haje venom was the least toxic in all assays. One ml of antivenom I neutralized 0.075 µg of svPLA2 in N. ashei venom compared to 0.051 µg by antivenom II. Antivenom I was ineffective in neutralizing N .ashei venom-induced lethality but 1 ml of antivenom II neutralized 0.21 mg of N. ashei venom.

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Comparison of a relatively toxic phospholipase A2 from Naja nigricollis snake venom with that of a relatively non-toxic phospholipase A2 from Hemachatus haemachatus snake venom—I. Enzymatic activity on free and membrane bound substrates

Toxicon ◽

10.1016/0041-0101(81)90054-4 ◽

1981 ◽

Vol 19 (3) ◽

pp. 439

Author(s):

G. Habermehl

Keyword(s):

Enzymatic Activity ◽

Phospholipase A2 ◽

Snake Venom ◽

Membrane Bound

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Mechanism of Inhibitory Action on Platelet Activation of a Phospholipase A2 Isolated from Lachesis muta (Bushmaster) Snake Venom

Thrombosis and Haemostasis ◽

10.1055/s-0038-1665414 ◽

1997 ◽

Vol 78 (05) ◽

pp. 1372-1380 ◽

Cited By ~ 56

Author(s):

André L Fuly ◽

Olga L T Machado ◽

Elias W Alves ◽

Célia R Carlinis

Keyword(s):

Platelet Aggregation ◽

Enzymatic Activity ◽

Phospholipase A2 ◽

Inhibitory Activity ◽

Thermal Inactivation ◽

Anticoagulant Activity ◽

Gel Filtration ◽

Apparent Molecular Weight ◽

Crude Venom ◽

Lachesis Muta

SummaryCrude venom from Lachesis muta exhibited procoagulant, proteolytic and phospholipase A2 activities. A phospholipase A2, denoted LM-PLA2 was purified from L. muta venom to homogeneity, through a combination of chromatographic steps involving gel-filtration on Sephacryl S-200 HR and reverse phase chromatography on a C2/C18 column. LM-PLA2 presented a single polypeptide chain with an isoelectric point at pH 4.7 and apparent molecular weight of 17 kDa. Partial aminoacid sequence indicated a high degree of homology for LM-PLA2 with other PLA2 from different sources.LM-PLA2 displayed a potent enzymatic activity as measured by indirect hemolysis of red blood cells but it was neither lethal when injected i.p. into mice nor did it present anticoagulant activity. Furthermore, LM-PLA2 displayed a moderate inhibitory activity on the aggregation of rabbit platelets induced by low levels of ADP, thrombin and arachidonate. In contrast, platelet aggregation induced by high doses of collagen was strongly inhibited by LM-PLA2 as well as ATP-release. Treatment of the protein with p-bromophenacyl bromide or 2-mercapto-ethanol, as well as thermal inactivation studies, suggested that the platelet inhibitory effect of LM-PLA2 is dependent on its enzymatic activity. Thus, the platelet inhibitory activity of LM-PLA2 was shown to be dependent on the hydrolysis of plasma phospholipids and/or lipoproteins, most probably those rich in phosphatidylcholine. Surprisingly, lyso-phosphatidylcholine released by LM-PLA2 from plasma was shown to preferentially inhibited collagen-induced platelet aggregation, in contrast to other PLA2s, whose plasma hydrolytic products indistinctly affect platelet’s response to several agonists.

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