Chun- Chang

The essentiality of calcium ion in the enzymatic activity of Taiwan cobra phospholipase A2.

In order to address the mechanism whereby Ca2+ wad crucial for the manifestation of the enzymatic activity of phospholipase A2 (PLA2), four divalent cations were used to assess their influences on the catalytic activity and the fine structures ofNaja naja atra PLA2. It was found that substitution of Mg2+ or Sr2+ for Ca2+ in the substrate solution caused a decrease in the PLA2 activity to 77.5% or 54.5%, respectively, of that in the presence of Ca2+. However, no PLA2 activity was observed with the addition of Ba2+. With the exception of Mg2+, the nonpolarity of the 8-anilinonaphthalene-1-sulfonate (ANS)-binding site of PLA2 markedly increased with the binding of cations to PLA2. In the meantime, the accessibilities of Lys-6 (65) and Tyr-3 (63) toward trinitrobenzene sulfonate andp-nitrobenzenesulfonyl fluoride were enhanced by the addition of Ca2+, Sr2+, and Ba2+, but not by Mg2+. The order of the ability of cations to enhance the ANS fluorescence and the reactivity of Lys and Tyr residues toward modified reagents was Ba2+> Sr2+> Ca2+> Mg2+, which was the same order as the increase in their atomic radii. These results, together with the observations that the ANS molecule binds at the active site of PLA2 and that Tyr-3, Lys-6, and Tyr-63 of PLA2 are involved in the binding with the substrate, suggest that the binding of Ca2+ to PLA2 induces conformational changes at the active site and substrate-binding site. However, the smaller atomic radius with Mg2+ or the bigger atomic radii with Sr2+ and Ba2+ might render the conformation improperly rearranged after their binding to PLA2 molecule.

Probing calcium ion-induced conformational changes of Taiwan cobra phospholipase A2 by trinitrophenylation of lysine residues.

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.

Chemical modification of cationic residues in toxin a from king cobra (Ophiophagus hannah) venom

The cationic groups of arginine and lysine residues inα-neurotoxin, Toxin a, isolated from king cobra (Ophiophagus hannah) venom were subjected to modification with trinitrobenzene sulfonate (TNBS) andp-hydroxyphenylglyoxal (HPG), respectively. The trinitrophenylated (TNP) derivatives of Toxin a at Lys-10, 56, or 71 showed approximately 25% residual lethality, and modifications on Lys-10 and 56 or Lys-10 and 50 resulted in a decrease of lethality by 84% and 86%, respectively. Modifications on Arg-34, 37, and 70 and Arg-34, 37, and 72 in Toxin a caused a decrease in lethality by 92% and 93%, respectively, and it almost completely lost its lethality and binding activity to nicotinic acetylcholine receptor (nAChR) when all four arginine residues were modified. These results indicate that in addition to the cationic residues on loop II (Arg-34, 37), loop III (Lys-50, 56), and the C-terminal tail (Arg-70, 72; Lys-71), Lys-10 on loop I is also related to the neurotoxicity of Toxin a.

Functional involvement of Lys-6 in the enzymatic activity of phospholipase A2 fromBungarus multicinctus (Taiwan banded krait) snake venom

Phospholipase A2 (PLA2) fromBungarus multicinctus snake venom was subjected to Lys modification with 4-chloro-3,5-dinitrobenzoate and trinitrobenzene sulfonic acid, and one major carboxydinitrophenylated (CDNP) PLA2 and two trinitrophenylated (TNP) derivatives (TNP-1 and TNP-2) were separated by high-performance liquid chromatography. The results of amino acid analysis and sequence determination revealed that CDNP-PLA2 and TNP-1 contained one modified Lys residue at position 6, and both Lys-6 and Lys-62 were modified in TNP-2. It seemed that the Lys-6 was more accessible to modified reagents than other Lys residues in PLA2. Modification of Lys-6 caused a 94% drop in enzymatic activity as observed with CDNP-PLA2 and TNP-1. Alternatively, the enzyme modified on both Lys-6 and Lys-62 retained little PLA2 activity. Either carboxydinitrophenylation or trinitrophenylation did not significantly affect the secondary structure of the enzyme molecule as revealed by the CD spectra, and Ca2+ binding and antigenicity of Lys-6-modified PLA2 were unaffected. Conversion of nitro groups to amino groups resulted in a partial restoration of enzymatic activity of CDNP-PLA2 to 32% of that of PLA2. It reflected that the positively charged side chain of Lys-6 might play an exclusive role in PLA2 activity. The TNP derivatives could be regenerated with hydrazine hydrochloride. The biological activity of the regenerated PLA2 is almost the same as that of native PLA2. These results suggest that the intact Lys-6 is essential for the enzymatic activity of PLA2, and that incorporation of a bulky CDNP or TNP group on Lys-6 might give rise to a distortion of the interaction between substrate and the enzyme molecule, and the active conformation of PLA2.

THE STRUCTURAL VARIATIONS OF EPSILON -AMINO GROUPS IN PHOSPHOLIPASE A2 ENZYMES FROM NAJA NAJA ATRA AND BUNGARUS MULTICINCTUS VENOMS

Comparative studies on Naja naja atra phospholipase A2 (NNA-PLA2), Bungarus multicinctus phospholipase A2 (BM-PLA2), and their Lys-modified derivatives were made to assess the differences in the fine structures around the conserved Lys residues of PLA2 enzymes. It was found that the accessibility of Lys residues of PLA2 enzymes toward modified reagent, trinitrobenzene sulfonate, were not the same. Moreover, the extent of decrease in pI values of PLA2 enzymes that resulted from trinitrophenylation of lysine residues was different between NNA-PLA2 and BM-PLA2. The Lys-6 of BM-PLA2 mostly contributed to the positively charged character of the enzyme molecule, whereas the contribution of Lys-6 of NNA-PLA2 to its molecular charge was not notably different from other Lys residues. A linear relationship was observed by plotting the mobilities of PLA2 enzymes and their TNP derivatives against their pI values. However, native and Lys-modified NNA-PLA2 were not aligned with those of BM-PLA2 in the same line. Apparently the gross conformation of PLA2 enzymes was not notably perturbed by the modification of Lys residues, but the fine structure of NNA-PLA2 was not the same as that of BM-PLA2. These results indicate that the positioning of side chains of the conserved Lys residues in the two PLA2 enzymes is essentially different, and suggest that the variations in the fine structures of homologous proteins could be effectively explored by chemical modification studies and electrophoretic analysis.

Chemical modification of tryptophan residues in alpha-neurotoxins from Ophiophagus hannah (king cobra) venom.

Twoα-neurotoxins, Oh-4 and Oh-7, from the king cobra (Ophiophagus hannah) venom were subjected to Trp modification with 2-nitrophenylsulfenyl chloride (NPS-Cl). One major NPS derivative was isolated from the modified mixtures of Oh-4 and two from Oh-7 by HPLC. Amino acid analysis and sequence determination revealed that Trp-27 in Oh-4, and Trp-30 and Trp-26 and 30 in the two Oh-7 derivatives, were modified, respectively. Sulfenylation of Trp-27 in Oh-4 caused about 70% drop in lethal toxicity and nicotinic acetylcholine receptor-binding activity. Modification of Trp-30 in Oh-7 resulted in the decrease of lethal toxicity by 36% and binding activity by 61%. The activities were further lost when the conserved Trp-26 in Oh-7 was modified. Sulfenylation of the Trp residues did not significantly affect the secondary structure of the toxins as revealed by the CD spectra. These results indicate that the Trp residues in these two longα-neurotoxins may be involved in the receptor binding.

The essentiality of B chain in stabilizing the structure of the A chain in β1-bungarotoxin fromBungarus multicinctus venom

The dynamic of Trp residue inΒ1-bungarotoxin (gb1-Bgt), the A chain ofΒ1-Bgt and phospholipase A2 (PLA2) was assessed by fluorescence measurement. Acrylamide quenching studies showed that the exposure degree of the Trp in PLA2 is higher than the Trp inΒ1-Bgt. The Trp ofΒ1-Bgt had a higher accessibility for iodide, reflecting that the basic nature of the B chain might exert an attractive electrostatic force for iodide and increase the susceptibility of Trp in the A chain to iodide. Removal of the B chain ofΒ1-Bgt did not significantly affect the exposure degree of Trp in the A chain. Alternatively, the polarity of the environment around the Trp and the hydrophobic character of ANS and substrate binding sites in the separated A chain changed. Measurement of Trp fluorescence with increasing temperature showed that the stability of structure ofΒ1-Bgt was higher than those of the separated A chain and PLA2. These results suggest that the B chain might interact with the A chain and stabilize the conformation of the A chain inΒ1-Bgt.

Probing the structural diversities of long alpha-neurotoxins by fluorescence quenching studies.

Trp fluorescence of Ophiophagus hannah neurotoxins (Oh-4, Oh-6A, Oh-7, and Oh-8) and Bungarus multicinctus α-bungarotoxin was quenched by acrylamide and iodide. Acrylamide quenching studies indicated that the degree of exposure of Trp residues in the neurotoxins followed the order Oh-8 > Oh-7 > Oh-6A > Oh-4 > α-bungarotoxin, as did the accessibility for iodide. These results reveal that the exposed degree of Trp residues and the microenvironment surrounding Trp residues in the neurotoxins differ, even though their Trp residues and positively charged residues are located at the same or homologous positions. In contrast to unfolded Oh-4, Oh-6A, Oh-7, and α-bungarotoxin, unfolding of Oh-8 by reduction and S-carboxymethylation caused a notable decrease in the susceptibility of their Trp residues for iodide. These observations support the view that the side chains of Trp residues and positively charged residues in their native structure do not point toward the same spatial positions. Computer models of the neurotoxins are in good agreement with this proposition. These results elucidate why the conserved Trp residues and cationic groups do not always play the same roles in the biological activities of the neurotoxins.

The structural and functional essentiality of the N‐terminal α‐helix in the phospholipase A2 of the Taiwan banded krait

In order to identify the structural and functional essentiality of the N‐terminal α‐helix of Bungarus multicinctus PLA2 for its enzymatic activity, comparative studies of the biochemical properties of native and recombinant PLA2 were made. It was found that the appearance of a Met residue preceding the N‐terminus Asn‐1 of the recombinant protein appreciably affected PLA2 activity and the hydrophobic character of the ANS‐binding site. Additionally, the charged state and the hydrophobicity of the molecular surface changed as well. However, removal of the N‐terminal Met‐1 from the recombinant PLA2 resulted in the production of a fully active PLA2, whose biochemical properties were indistinguishable from those of the native enzyme. These observations, together with the findings that the helical wheel plot of the N‐terminal α‐helix showed distinct hydrophobic and hydrophilic faces and that the ANS‐binding site was the active site of PLA2 enzymes, suggest that the hydrophobic face of the N‐terminal α‐helix in native PLA2 should be in the interior of the enzyme molecule for binding with ANS and the phospholipid/substrate.

THE INTERACTION OF 8-ANILINONAPHTHALENE-1-SULFONATE WITH HIS-47 OF TAIWAN COBRA PHOSPHOLIPASE A2 PERTURBING BY THE BINDING OF CALCIUM ION

The 8‐anilinonaphthalene‐1‐sulfonate (ANS) fluorescence intensity of ANS‐Naja naja atra (Taiwan cobra) phospholipase A2 (PLA2) complex increased with the addition of Ca2+, but the observed fluorescence enhancement markedly decreased after methylation of His‐47 in PLA2 molecule. However, the binding affinities of methylated PLA2 for ANS and Ca2+ were similar to or even greater than those observed with native PLA2. These results, together with the finding that ANS electrostatically interacted with His‐47 of PLA2, suggest that the increase in the intensity of ANS fluorescence upon the addition of Ca2+, in part, arises from the ionic interaction of His‐47 with ANS being perturbed by the binding of Ca2+.