Preparation and Characterization of an Immuno-electron Microscope Tracer Consisting of a Heme-octapeptide Coupled to Fab

A heme-octapeptide (mol wt 1,550) has been obtained from cytochrome c by successive pepsin and trypsin hydrolysis and purified by gel filtration and countercurrent distribution. It possesses peroxidatic activity characterized by an apparent K(m) of 0.2 M, an apparent v(max) of 4 mmol/min per mg of peptide, and a pH optimum of 7.0. Using a novel two-step conjugation procedure, the heme-octapeptide was coupled to rabbit Fab antibody fragments by first derivatizing it with the N-hydroxysuccinimide ester of p-formylbenzoic acid and subsequently allowing it to form a Schiff base with the amino groups of Fab. Stable covalent linkages were then obtained by reduction of the Schiff bases with sodium borohydride. The conjugate consists of approximately 2 heme-octapeptides attached to each Fab molecule. The molecular weight is 45,000 daltons when coupled to sheep Fab and 50,000 daltons with a Stokes radius of 32 A, when conjugated to rabbit Fab. Its peroxidatic activity is characterized by an apparent K(m) of 0.4 M, an apparent v(max) of 0.4 mmol/min and per mg of attached heme-octapeptide and a pH optimum of 7.0. The conjugate has been used for the localization at the electron microscope level of secretory immunoglobulins in the mammary gland of lactating rabbits.