Localization of angiotensin converting enzyme (Kininase II). I. Preparation of antibody heme octapeptide conjugates

Antibodies to pig lung angiotensin converting enzyme (kininase II) were conjugated to a heme octapeptide (8 microperoxidase, 8 MP) derived from cytochrome c. 8 MP, which has only one reactive amine, was coupled to antibody in a twostep procedure using a bifunctional active ester, bis succinyl succinate. In the first step, 8 MP was reacted with an excess of bis succinyl succinate to yield 8 MP succinyl succinate, a stable compound which can be stored. In a second step the remaining active ester was used for coupling to reactive amines of the antibody. The conjugate consists of 1.6-2.3 8 TP moieties per antibody. Using these procedures, the formation of complex polymers is avoided. Each molecular of conjugate possesses both immunoreactivity and peroxidatic activity. The conjugate has been used to localize angiotensin converting enzyme along the plasma membrane and associated caveolae of pig aortic endothelial cells in culture.