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 two-step 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-MP moieties per antibody. Using these procedures, the formation of complex polymers is avoided. Each molecule of conjugate possesses both immuno-reactivity 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.