Carbamylation with cyanate of basic phospholipase A 2 from the venom of Naja nigricollis (Spitting cobra)

Abstract

The most basic and toxic phospholipase A 2 purified from Naja nigricollis venom was subjected to lysine modification with cyanate at pH 8.0. The carbamylated derivatives were separated by chromatography on a column of SP-Sephadex C-25 and five fractions were obtained. Amino acid analysis showed that the numbers of Lys-residues modified for fractions Fa to Fe were 10, 9.2, 9.0, 8.2 and 7.4 respectively. The pI values decrease with increasing carbamylation, converting the basic enzyme into an acidic protein. It is noteworthy that even after 9 out of 10 Lys-residues had been modified (Fc) and the pI of the enzyme decreased from 10.6 to 4.4, the enzyme still possessed 75% of its enzymatic activity and 51% of its antigenicity. Although both native and Lys-modified N. nigricollis phospholipase A 2 were perturbed by the presence of Ca 2+, the difference spectra of Lys-modified Fd differ greatly from that of the native enzyme and become similar to that of the acidic and less toxic phospholipase A 2 from N. naja atra venom. At pH 8.0, the effect of Ca 2+ on the fluorescence emission intensity of 8-anilinonaphthalenesulfonate-Lys-modified Fd complex was different from that of 8-anilinonaphthalenesulfonate-native N. nigricollis phospholipase A 2 complex and was similar to that of 8-anilinonaphthalenesulfonate- N. naja atra phospholipase A 2 complex. It is clear that the different conformational changes induced by Ca 2+ might be attributable to the charge properties of the enzyme, which depend on the pH of the solution and the pI of the enzyme. These data are discussed in relationship to the decreased lethal potency and pharmacological effects induced by carbamylation ( Condrea et al., 1981b).