A Specific Method for Purification of the Second Component of Guinea Pig Complement and a Chemical Evaluation of the One-Hit Theory

Abstract

The second component of guinea pig complement (C2) may be isolated in good yield and in highly purified form by a specific method involving adsorption on EAC4 (sheep erythrocytes carrying rabbit anti-Forssman and guinea pig C4) at ionic strength 0.0686, pH 8.5 and 25° to 30°C, in the presence of 0.002 M MgCl2, 0.0003 M CaCl2 and 0.1% gelatin, followed by elution at ionic strength 0.147, pH 6.5 and 0°C, in the presence of 0.05% gelatin. The product is stable and does not contain guinea pig serum proteins other than C2, but there is heavy contamination with sheep erythrocyte proteins, mostly hemoglobin, which may be removed by two successive electrophoretic fractionations on polyacrylamide gel. The number of C2 molecules required for lysis of one cell was 1.1.