Early events of pepsinogen activation

Stopped-flow measurements both with native pig pepsinogen and with a fluorescent derivative, labeled near the carboxyl terminus with a toluidinylnaphthalenesulfonyl (TNS) group at Lys364, show rapid fluorescence changes following acidification. The rate constants observed by intrinsic fluorescence of the native zymogen are distinctly greater than those exhibited by the TNS derivative in the pH range examined. The rate constants for two early events in the activation of the derivative increase as the pH decreases from pH 3 to pH 2. The fluorescent intensities of these two processes also vary with pH. Because the ratios of these amplitudes fit the Henderson-Hasselbalch equation, it is concluded that the two processes represent concurrent events, rather than sequential ones. It is proposed that a protonation separates two forms of the zymogen. The conjugate acid undergoes the slower event, whereas the conjugate base, which predominates at pH 3, undergoes the faster event. It is proposed that both these pathways result in activation.