Two
phospholipases A2 (PLA2) (EC 3.1.1.4) were purified from Bothrops neuwiedii
venom (
isoenzymes P-1 and
P-2). The molecular weights of P-1 and
P-2 as estimated by
sodium dodecyl sulfate-
polyacrylamide gel electrophoresis were 15,000 and 16,200 and the isoelectric points were 4.8 and 4.6, respectively. The N-terminal 14-amino-acid sequences determined were Asn-Leu-Val-Gln-
Phe-Glu-Thr-Leu-Ile-
Met-Met-Ile-
Ala-Gly and Ser-Leu-Val-Gln-
Phe-Glu-Thr-Leu-Ile-
Met-Met-Ile-
Ala-Gly for P-1 and
P-2, respectively. Since both show sequence almost identical to that of a PLA2 from Crotalus atrox it was tentatively classified as being of group II. The enzymatic activity of P-1 and
P-2 toward
lipid monolayers was studied. The hydrolysis of
dilauroylphosphatidylcholine (dlPC) shows a broad optimum between 7 and 18 mN m-1 and a cut-off pressure of 22 mN m-1. The activity toward dlPC displays a maximum at pH 8 and is dependent on the presence of Ca2+ with an apparent Kd of 0.1 mM, for both
enzymes. P-1 and
P-2 are heat-stable
enzymes, unable to hydrolyze
dilauroylphosphatidic acid monolayers. The
enzymes are not lethal to mice at doses up to 5 micrograms/g
body weight by
intraperitoneal injection and they do not show myotoxic (up to 40 micrograms) or hemolytic activity (up to 8.5 micrograms/ml). Both lack anti-
coagulant activity, determined by absence of changes in the recalcification time of platelet poor plasma (up to 100 micrograms/ml), and are not able to induce platelet aggregation (up to 50 micrograms/ml). However, both
isoenzymes exhibit an important
edema-inducing activity that is not altered at short times by the irreversible chemical inactivation of the hydrolytic activity with
phenacyl bromide. P-1 and
P-2 are able to release
arachidonic acid from membrane
phospholipids of neutrophils, property that is lost by the inactivation of the
enzyme. This suggests that the
edema-inducing activity of the active but not the inactive forms may be partly due to
arachidonic acid-derived mediators. The
edema-inducing activity of the active or inactive forms of the
enzymes is inhibited by antagonists of
histamine, suggesting that
histamine plays an important role in both the active and the inactive B. neuwiedii PLA2s-induced
edema. The results suggest that the inflammatory and the catalytic activity of these
enzymes constitute separate properties.