Proton Nuclear Magnetic Resonance Characterization of Phospholipase A2 from Laticauda semifasciata

• Published in: Journal of biochemistry (Tokyo) Vol. 101; no. 3; pp. 795 - 804
• Main Authors: ENDO, Toshiya, OYA, Masanao, TAMIYA, Nobuo, MIYAZAWA, Tatsuo
• Format: Journal Article
• Language: English
• Published: Oxford Oxford University Press 01.03.1987
• Subjects: Analytical, structural and metabolic biochemistry; Animals; Biological and medical sciences; Calcium; Circular Dichroism; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Histidine; Hydrogen-Ion Concentration; Hydrolases; Magnetic Resonance Spectroscopy; Molecular Weight; Phospholipases; Phospholipases A; Phospholipases A2; Snakes; Spectrometry, Fluorescence; Spectrophotometry, Ultraviolet; Temperature; Enzyme; Dissociation constant; NMR spectrometry; Phospholipase A; Ophidia; Marine environment; Vertebrata; Animal; Venom; Proton; Reptilia; Physicochemical properties; Conformational transition
• ISSN: 0021-924X; 1756-2651
• DOI: 10.1093/jb/101.3.795

The molecular properties of phospholipases (PLases) A2 I and A2 III from a sea snake, Laticauda semifasciata, have been characterized by gel-filtration, as well as proton NMR, CD, UV absorption, and fluorescence spectroscopic methods. PLase A2 I exists as a monomer in aqueous solution in the presence or in the absence of Ca2+ The dissociation constants of the Ca2+ complexes have been determined for the two enzymes. The 270-MHZ proton NMR spectra of PLases A2 I and A2 III have been measured, and the aromatic proton resonances of His-21 and His-48 in the active site have been assigned. By analyzing the pH dependence of the chemical shifts of the histidine proton resonances, pKa values have been deter mined for His-21 and His-48 with and without Ca2+ The conformational transitions have been found to take place at low pH or at high temperature (at ~65°C). Fluorescence change of PLase A2 I upon addition of substrate analogs suggests that Trp-70 in PLase A2 I is involved in the binding to micellar substrates. The lack of Trp–70 in PLase A2 III is probably related to the low enzymatic activity as compared with that of PLase A2 I.