Ferreira J P, Sasisekharan R, Louie O, Langer R
Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge 02139.
Biochemistry. 1993 Aug 17;32(32):8098-102. 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.
用两种不同的化学方法将眼镜蛇毒中的磷脂酶A2共价偶联到琼脂糖珠上。评估了偶联介质中胶束竞争性抑制剂的作用。与N-羟基琥珀酰亚胺活化的琼脂糖结合的酶，其主要对ε-氨基有反应性，对胶束二庚酰磷脂酰胆碱（DiC7-PC）的活性保留率为20%。通过改良的碳二亚胺法通过羧基结合的酶，活性保留率为50%。对于这两种偶联物，在单体二己酰磷脂酰胆碱以及Triton X-100与二棕榈酰磷脂酰胆碱或二油酰磷脂酰乙醇胺的混合胶束中观察到了相似的相对活性。酶的可溶形式对单体DiC7-PC表现出前胶束活化作用，而固定化形式在临界胶束浓度附近的浓度下表现出界面识别作用。这些结果表明，固定化后酶活性的丧失是酶固有化学修饰的结果，并且酶的寡聚化和界面识别不是因果现象。
