Activation of spin-labeled chicken pepsinogen

Chicken pepsinogen has been spin-labeled by the attachment of four nitroxides to epsilon-amino groups near the protein's amino terminus. Acidification results in a bond cleavage, generating a nonlabeled, enzymatically active protein. Electron spin resonance spectra of the spin-labeled zymogen, acidified in the presence or absence of pepstatin, are identical and indicate that the nitroxides are quite mobile, compared to the nonacidified zymogen. This mobilization is interpreted as the freeing of the peptide to which the spin-labels are attached, from the protein, subsequent to the acidification that causes a peptide bond cleavage. The rate at which the peptide leaves the protein is 1 order of magnitude slower than the cleavage of the peptide bond, measured by the rate of appearance of milk-clotting activity (first-order rate constants of 0.3 min-1 vs. 6 min-1 at pH 2, 22 degrees C). The inclusion of pepstatin, at molar ratios above 2 during activation, decreases the rate of peptide leaving. These observations, and those previously reported for activation of spin-labeled pig pepsinogen, are incorporated into a model of pepsinogen activation.