[Affinity chromatography for purification of phospholipase A2 from the venom of Central Asian cobra]. / Affinnaia khromatografiia dlia ochistki fosfolipazy A2 iada sredneaziatskoi kobry.

A method of affinity chromatography was developed for purification of phospholipase A2(PL-A2) from the Central Asian cobra venon. The enzyme was covalently coupled to a polyamide sorbent with phosphatidilethanolamine (PEA) and cytotoxin (CT). The effect of CA2+ concentration and the ion strength of the solution on the enzyme adsorption was studied. The most efficient coupling of the enzyme to the sorbent was observed at pH 8--9 in case of the Ca2+ absence and a low ion strength of the solution. For desorption of the enzyme Triton X-100 at a concentration of 0.5% should be introduced in the eluting solution. The affinity adsorption chromatography enabled the isolation of two forms of phospholipase A2 with different affinity for PEA and CT. The total yield of the enzyme was 91% at a purification degree of 5.5 and 3.5, respectively. The introduction of the second ligand (CT) in the composition of the sorbent with the phospholipid ligand allowed the authors to increase its capacity and affinity for the phospholipase A2 from the snake venom.

[Immobilization of phospholipase A2 from Central Asian cobra venom on polyamide sorbents]. / Immobilizatsiia fosfolipazy A2 iada sredneaziatskoi kobry na sorbentakh poliamidnoi prirody.

The effect of the immobilization technique and the ligand nature on catalytic properties of phospholipase A2 from the cobra venom was studied. Preparations of phospholipase A2 adsorbed on and covalently bound to polyamide sorbents were obtained. The enzyme was coupled to polyamide beads modified with glutaraldehyde. In this case only 9% of the enzyme activity was retained. The enzyme adsorbed on polyamide modified with phosphatidylethanolamine retained up to 20% of the initial activity. The binding selectivity of phospholipase A2 was maximum in case of the sorbent with a binary ligand, e. g. phosphatidylethanolamine+cytotoxin, the sorbent capacity for the bound enzyme increased 2-3 times (460-600 units/g sorbent. The specific activity of the adsorbed phospholipase A2 was 17-40 units/g sorbent in contrast to 8.6 units/g sorbent for the covalently bound enzyme. Immobilization of the enzyme on polyamide sorbents resulted in changes of the pH-optimum, sensitivity to Ca2+ ions and the character of the enzyme-substrate interactions. Heart stability of the adsorbed phospholipase A2 was lower than that of the covalently bound enzyme. However, the adsorbed enzyme can be used, for example, in affinity chromatography due to its higher specific activity, selectivity and reversibility of the sorption.

Structural changes of liposome phospholipid packing induced by cytotoxin of the Central Asia cobra venom.

Liposomes and proteoliposomes obtained from rat brain were used; structural changes induced by Vc5 cytotoxin (CT) from Central Asia cobra venom have been studied by the EPR method using spin probes (5-, 10-, or 12-doxylstearic acid). The addition of CT to liposome samples, containing spin probes resulted in the appearance of a new EPR signal in the initial spectrum (samples without CT), typical of probes with strongly retarded mobility. The presence of hydrophobic interaction between the CT molecules and spin labelled fat acids permits the assumption that CT molecules in liposomes trap both lipid probes and phospholipids localized in the reach of action of hydrophobic forces. CT may be supposed to induce formation in membranes of liposomes with domain structures. As a result of hydrophobic interaction with CT molecules both the phospholipid and lipid probe mobility in the domain is substantially less than that in liposome regions free of CT molecules. Due to this, a new signal appears in the initial EPR spectrum of the spin probes. An analysis of the dependence of the probe order parameter value on CT concentration in samples has suggested that CT act uniformly along the membrane lipid profile with a certain CT concentration range. At high concentrations CT molecules cannot penetrate the lipid region deep enough, due to mutual electrostatic repulsion and steric factors at membrane surface. As a result, structural changes involve regions adjacent to the membrane surface only.