Studies on the status of lysine residues in phospholipase A2 from Naja naja atra (Taiwan cobra) snake venom.
Phospholipase A2 (PLA2) from Naja naja atra (Taiwan cobra) snake venom was subjected to lysine modification with trinitrobenzene sulphonic acid (TNBS), and two major trinitrophenylated (TNP) derivatives, TNP-1 and TNP-2, were separated by h.p.l.c. TNP-1 contained only one TNP group on Lys-6 and showed a marked decrease in enzymic activity, but still retained 45% of the lethal toxicity. Both Lys-6 and Lys-65 were modified in TNP-2, and modification of Lys-65 caused a further reduction of the lethal toxicity to 12.6%. However, the antigenicity of both TNP-1 and TNP-2 remained unchanged. The reactivity of Lys-6 and Lys-65 toward TNBS was greatly enhanced by Ca2+ and dihexanoyl-lecithin, suggesting that the two Lys residues are not directly involved in the binding of Ca2+ and substrate. The modified derivatives retained their affinity for Ca2+, indicating that Lys-6 and Lys-65 did not participate in the Ca2+ binding. The TNP derivatives could be regenerated with hydrazine hydrochloride. The biological activities of the regenerated PLA2 are almost the same as those of native PLA2. These results indicate that Lys-6 and Lys-65 are important for the biological activities of PLA2, and incorporation of a bulky TNP group on Lys-6 and Lys-65 might give rise to a distortion of the active conformation of PLA2.
Yang
C C
CC
Institute of Life Sciences, National Tsing Hua University, Hsinchu, Taiwan, Republic of China.
Chang
L S
LS
D000818:Animals; D002118:Calcium; D002851:Chromatography, High Pressure Liquid; D004546:Elapid Venoms / Q000032:analysis*; D008239:Lysine / Q000032:analysis*; D010740:Phospholipases / Q000032:analysis*; D010741:Phospholipases A / Q000032:analysis*; D054467:Phospholipases A2; D011487:Protein Conformation; D014302:Trinitrobenzenesulfonic Acid
D004546:Elapid Venoms; D014302:Trinitrobenzenesulfonic Acid; D010740:Phospholipases; D010741:Phospholipases A; D054467:Phospholipases A2; D008239:Lysine; D002118:Calcium