Purification and properties of the C4 inactivator from nurse shark serum

Abstract

Highly purified C4 inactivator (C4I) has been isolated from a crude C4I preparation. The crude C4I activity was generated from nurse shark serum by the procedure of Jensen (1969). The procedures employed in the purification were; (A) ammonium sulfate fractionation of the crude C4I, (B) DE 52 column chromatography, (C) hydroxyapatite column chromatography, (D) Sephadex G-100 gel filtration and (E) preparative acrylamide gel electrophoresis. Analytical disc gel electrophoresis at steps A through D in the purification procedure revealed five closely spaced protein bands (three major and two minor) that exhibited C4I activity. The five heteromorphs were separated at Step (E). Rabbit antiserum against crude C4I gave single line and arc on ouchterlony and immunoelectrophoresis with the purified material (D). Moreover, antiserum against D gave a single arc with the crude C4I. The five isomers showed esterase activity of similar specificity with Nα-acetylglycyl- l-lysine methyl ester and Nα-tosyl- l-arginine methyl ester. Experimental evidence indicates that the five heteromorphs are charge isomers. A mol. wt of ca. 67,000 was calculated for the isomers by analytical gel electrophoresis and of 70,000 by Sephadex gel filtration. The minimum mol. wt by SDS gel electrophoresis was 23,000. A three subunit structure for C4I is suggested. Treatment of human C4 with purified C4I at 37°C for 30 min resulted in a cleavage of the C4 molecule.