Abstract
Phospholipase A2 (PLA2) from Naja naja atra (Taiwan cobra) snake venom was subjected to lysine modification with trinitrobenzene sulfonate (TNBS). Three major derivatives, TNP-1, TNP-2, and TNP-3, were separated by high-performance liquid chromatography (HPLC) from the reaction mixtures in the absence of Ca2+. However, only TNP-2 and TNP-3 were isolated when trinitrophenylated reaction was carried out in the presence of Ca2+. TNP-1 and TNP-2 contained only one TNP group, on Lys-65 and Lys-6, respectively; and both Lys-6 and Lys-65 were modified in TNP-3. The extent of modification on Lys-6 and Lys-65 was calculated from the peak areas of TNP proteins in the HPLC profile. It was found that the susceptibility of Lys-6 toward TNBS markedly increased by the addition of Ca2+ when Ca2+ concentration was higher than 5 mM. With regard to the involvement of Lys-6 in the binding of substrate, the increase in the reactivity of Lys-6 may arise from a conformational change around Lys-6 for binding with substrate in the presence of Ca2+. Alternatively, the nonessentiality of Lys-65 for PLA2 activity was revealed by the finding that TNP-1 still retained 95% activity of native enzyme. Moreover, the reactivity of Lys-65 toward TNBS did not greatly change in either the absence or presence of Ca2+, suggesting that Ca2+ binding did not cause an appreciable change in the microenvironment around Lys-65. These results indicate that the differential reactivities of Lys-6 and Lys-65 toward TNBS as affected by the binding of Ca2+ are well consistent with their functional roles in the catalytic mechanism of PLA2, and suggest that the occurrence of conformational changes with PLA2 could be explored by chemical modification studies.
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Chang, Ls., Lin, Sr. & Chang, Cc. Probing Calcium Ion-Induced Conformational Changes of Taiwan Cobra Phospholipase A2 by Trinitrophenylation of Lysine Residues. J Protein Chem 16, 51–57 (1997). https://doi.org/10.1023/A:1026342928175
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DOI: https://doi.org/10.1023/A:1026342928175