A Single Scheme for C3a and C5a Isolation and Characterization of Chemotactic Behavior

Abstract

Abstract A four-step procedure was utilized in the isolation of two complement-derived anaphylatoxins (C3a and C5a) from yeast cell activated human serum. Advantage was taken of one gel filtration and three ion exchange chromatographic steps to obtain two homogeneous products. The carboxypeptidase inhibitor epsilon-aminocaproic acid (EACA) was added to the serum to prevent inactivation of the anaphylatoxins during their generation. Purified C3a and C5a were judged to be homogeneous by sodium dodecyl sulfate polyacrylamide gel electrophoresis (PAGE), acid PAGE, and cellulose acetate strip electrophoresis. A single precipitin are was detected when each agent was challenged with the corresponding rabbit antibody after immunoelectrophoresis. Recovery of the respective anaphylatoxins from 1 liter of activated serum was 6 to 8 mg for C3a and 400 to 600 µg for C5a. A partial chemical characterization of the C5a revealed that arginine was released from the COOH-terminus by carboxypeptidase B digestion. Arginine, leucine, and glycine were released by yeast carboxypeptidase. Amino acid analysis of human C5a indicates a gross similarity between human C5a and porcine C5a, but not between human C3a and human C5a. This current scheme for isolating human C3a and C5a by a single procedure represents a major advance in obtaining highly purified anaphylatoxins. The methodology should prove immediately applicable for isolation of both anaphylatoxins from the sera of animals. The minimal dose for smooth muscle contraction were 350 to 500 ng for C3a and 50 to 75 ng for C5a. Pure human C3a and C3 as were inactive for inducing chemotaxis with human PMN over a range of 10−5 to 10−10 M. C5a was chemotactically active between 3 × 10−10 M and 1.5 × 10−8 M whereas C5a1 was only marginally active. However, when quantities of C5a1 are added to normal human serum at a level comparable to that in complement activated serum, chemotactic activity is observed, suggesting C5a1 is a major factor in the generation of leukocyte-stimulating activity in human serum.