(A) study on the disulfide bonds of guamerin, a protease inhibitor from a Korean native leech

Abstract

Guamerin, a humam leukocyte elastase inhibitor from the Korean leech Hirudo nipponia is composed of 57 amino acid residues with ten cysteines. Disulfide bonds between two cystein residues were analyzed by biochemical methods. Thermolysin digestion of guamerin revealed that it had two domains containing two and three disulfide bonds, respectively. The N-terminal domain was revealed to have Cys1-Cys3 bond(disulfide bond between the first cysteine and the third cysteine) and Cys2-Cys4 bond. The disulfide bond pattern of the C-terminal domain was one of ``Cys5-Cys8, Cys6-Cys9 and Cys7-Cys10`` or ``Cys5-Cys8, Cys6-Cys10 and Cys7-Cys9`` patterns, that was not completely determined yet. Among the five disulfide bonds, there were two weak disulfide bonds more easily modified by 4-vinylpyridine at pH8.0. Likewise, recombinant guamerin expressed in the yeast Pichia pastoris and guamerin II, a serine protease inhibitor from another Korean leech, Whitmania edentula were revealed to have identical disulfide bond pattern and characteristics with guamerin. Furthermore, their disulfide bond patterns were consistent with those of hirustasin and antistasin, another serine protease inhibitors from other leech species, of which three dimensional structures were already determined by X-ray crystallography.