Interactions of Trimeresurus flavoviridis Phospholipase A2 and Its N-Terminal Octapeptide-Removed and p-Bromophenacylated Derivatives with Acridine and Anilinonaphthalene Dyes

Abstract

Interactions of dimeric Trimeresurus flavoviridis (the Habu snake) phospholipase A2 (PLA2), des-octapeptide(1-8)-PLA2 (L-fragment) (14% of PLA2 activity), and p-bromophenacyl bromide (BPB)-inactivated PLA2 (BP-PLA2) with dyes, namely, proflavine, 1-anilinonaphthalene-8-sulfonate (Ans), and 2-toluidinylnaphthalene-6-sulfonate (Tns), were investigated. All dyes were bound in a 1:1 molar ratio to the subunit of the proteins. Proflavine was bound most strongly to PLA2 and Ans and Tns were bound to the three proteins with comparable affinities. Capabilities of the dyes for inhibiting alkylation of His-47 of PLA2 with BPB were in the following order: Ans greater than proflavine greater than Tns. Fluorescences of Ans and Tns that were increased in the presence of PLA2 were further greatly enhanced upon the addition of Ca2+, with concomitant formation of the ternary complexes. Ca2+, however, inhibited, competitively or noncompetitively, the bindings of the dyes to PLA2. All dyes were bound to the active site of PLA2 but with different orientations. Inactivation of L-fragment with BPB was inhibited by the dyes in the following order: Tns greater than proflavine approximately Ans. Addition of Ca2+ to the binary complexes formed from L-fragment and Ans or Tns caused no additional enhancement of fluorescence in spite of the formation of the ternary complexes. The active site structures are different between PLA2 and L-fragment, and the N-terminal octapeptide moiety of PLA2 possibly plays a role in maintaining the optimally arranged active site structure of the molecule. Comparison of the data suggests that the N-terminal moieties of PLA2S from snakes of an elapid family and from mammalian pancreas are essential for catalysis of a micellar substrate, whereas those of PLA2S from snakes of a viperid family, such as T. flavoviridis, are not. BP-PLA2 bound Ca2+ and was similar to L-fragment in terms of the fluorescence measurements. It appears that the active site of PLA2 has a space large enough to accommodate p-bromophenacyl, Ans or Tns, and Ca2+ together. Comparison of the emission maxima of Ans and Tns complexed with the three proteins indicated that Tns could be a useful fluorescent probe informing us of the state (disorder) of the active site of PLA2.

Heparin prevents the binding of phospholipase A2 to phospholipid micelles: importance of the amino-terminus

“…Heparin Influences the Structure of B-PLA2. ANS binds to PLA2 in the vicinity of the active site (Oda et al, 1986;van Eijk et al, 1984). The heparin-induced change in ANS/B-PLA2 fluorescence suggests that heparin influences the active site.…”

Purification and characterization of a novel phospholipase A2 from king cobra (Ophiophagus hannah) venom

“…Thus it was of interest to see whether the two conserved residues were involved in the enzymatic activity of Oh-DE-2. The catalytic activity of Oh-DE-2 was completely lost after reaction with 100-fold molar excess of p-bromophenacyl bromide for 8hr, indicating that the His-48 was closely related to its enzymatic activity, as in other PLAzs from snake venoms (Kondo et al, 1981a;Oda et aL, 1986;Yang and King, 1980;Yang et al, 1981). Alternatively, Oh-DE-2 contains only one Trp residue at position 19 as revealed by Nbromosuccinimide titration .and amino acid sequence.…”

Differences in primary structure among five phospholipases A2 from Heloderma suspectum

“…A hydrogen-bonding network links the N-terminus in pancreatic and C. atrox enzymes to the catalytic center, thereby facilitating a proper orientation and penetration of the substrate present at the surface of a detergent micelle, such as deoxycholate or Triton X-100 [8,10,11,13,141. The Nterminal interface recognition site includes Gln4, Ile9, Cys45 and Tyr69 in bovine phospholipase A, [8], and the removal of the N-terminal octapeptide from the porcine and elapid enzymes abolishes all enzymatic activity against micellar substrates [15, 161. In the present lizard variants Pal -Pa,, as in the honeybee enzyme, the short and highly hydrophobic N-terminal extremity was severely curtailed, and a strategic Gln4 residue 1171 was absent.…”