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. 1974 Jan 1;139(1):208–223. doi: 10.1084/jem.139.1.208

PREPARATION AND CHARACTERIZATION OF AN IMMUNOELECTRON MICROSCOPE TRACER CONSISTING OF A HEME-OCTAPEPTIDE COUPLED TO Fab

J P Kraehenbuhl 1, R E Galardy 1, J D Jamieson 1
PMCID: PMC2139508  PMID: 4357686

Abstract

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 Km 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 ∼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 Å, when conjugated to rabbit Fab. Its peroxidatic activity is characterized by an apparent Km 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.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

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