Influence of chemistry in immobilization of cobra venom phospholipase A2: Implications as to mechanism

• Published in: Biochemistry (Easton) 1993-08, Vol.32 (32), p.8098-8102
• Main Authors: Ferreira, Joao Paulo M, Sasisekharan, Ram, Louie, Otway, Langer, Robert
• Format: Article
• Language: English
• Subjects: Binding, Competitive; Biological and medical sciences; Biotechnology; Elapid Venoms - chemistry; Enzyme Activation; Enzymes, Immobilized; Fundamental and applied biological sciences. Psychology; Immobilization of enzymes and other molecules; Immobilization techniques; Methods. Procedures. Technologies; Micelles; Octoxynol; Phosphatidylcholines - metabolism; Phospholipases A - antagonists & inhibitors; Phospholipases A - chemistry; Phospholipases A - metabolism; Phospholipases A2; Phospholipids - metabolism; Polyethylene Glycols; Protein Binding; Sepharose; Succinimides
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00083a007

Phospholipase A2 from Naja naja kaouthia venom was covalently coupled onto agarose beads using two different chemistries. The effect of micellar competitive inhibitors in the coupling media was evaluated. Enzyme bound to N-hydroxysuccinimide-activated agarose, which is reactive primarily toward epsilon-amino groups, had 20% activity retention against micellar diheptanoylphosphatidylcholine (DiC7-PC). Enzyme bound through carboxylic groups, using a modification of the carbodiimide method, had 50% retention. Similar relative activities were observed, for both conjugates, in monomeric dihexanoyl-PC and in mixed micelles of Triton X-100 with dipalmitoyl-PC or dioleoylphosphatidylethanolamine. The soluble form of the enzyme showed premicellar activation against monomeric DiC7-PC, while the immobilized form showed interfacial recognition at concentrations around the critical micellar concentration. These results suggest that the enzyme activity lost upon immobilization is a result of the inherent chemical modification of the enzyme and that enzyme oligomerization and interfacial recognition are not cause-effect phenomena.