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Further Studies on the Effect of Ca⁺⁺ on the First Component of Human Complement (C'1)¹

From the Institute of Pathology, Western Reserve University, Cleveland, Ohio

Abstract

The kinetics of inactivation of C'1 in human serum treated with Na₃ EDTA are reported. The extent of reaction was dependent on time and temperature but was insensitive to pH and ionic strength. Complete inactivation of C'1, following first order kinetics after an initial accelerated phase, was achieved by incubating serum at unadjusted pH and ionic strength with 10^-2 to 5 x 10^-2 M Na₃ EDTA at 45°C for 4–5 hr. The specific effect of Ca⁺⁺ in stabilizing C'1 was shown by using MgNa EDTA and CaNa EDTA under the same conditions. Although partial inactivation of other components of complement occurred in some experiments, the primary effect of Ca⁺⁺-depletion was on C'1. The activity of C'1 could not be restored after complete inactivation had occurred but partial restoration was possible after partial inactivation.

Sera depleted of C'1 by treatment with Na₃ EDTA (R1-EDTA) were tested as reagents for measurement of C'1 in a human serum pool and in a purified fraction. When prepared under carefully controlled conditions of concentration of Na₃ EDTA, temperature, time, and ratio of surface area to serum volume, R1-EDTA compared favorably with conventional R1 prepared by dialysis as a reagent for such titrations.

Footnotes

This investigation was supported by grants from the United States Public Health Service (Pathology Training Grant #HST-5137, and Institute of Allergy and Infectious Diseases, Grant #E-1255c) and from the Commission on Immunization, Armed Forces Epidemiological Board.

² United States Public Health Service Training Fellow in Pathology.

³ Research Fellow of the Cleveland Area Heart Society.