Immobilization of proteins as a tool for studying primary structure around their cysteinyl residues

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Abstract
The primary structure around the single cysteinyl residue of chicken pepsin was investigated by binding the protein via this residue to an insoluble carrier. Carriers stable towards reagents used for the fragmentation of proteins and sequence analysis were prepared by coupling a spacer arm to polyN-hydroxymethyl acrylamide using a thioether bond that is potentially cleavable by mercuric ions (1). Phenacyl bromide group, attached to the free end of the spacer, reacted rapidly and specifically with the cysteinyl residue of chicken pepsin. Up to 300 mg of the enzyme were bound to 1 g of carrier. The polymer-bound protein was cleaved by trypsin or by cyanogen bromide or by a sequence of both. Fragments of 40–120 amino acid residues, depending on the method of cleavage, remained attached to the polymer through the cysteinyl residue. The compositions and partial sequences of these fragments revealed that the cysteinyl residue is located within or in the vicinity of a loop in the molecule formed by a disulfide bond.