Prochymosin, the zymogen of the calf milk-clotting enzyme chymosin, has recently been cloned and expressed in Escherichia coli (Harris et al., 1982). Like many animal secretory proteins which have been expressed at high levels in E. coli, recombinant prochymosin aggregates and forms refractile granules within the bacteria (Emtage et al., 1983). After recovery and purification from these granules, recombinant prochymosin can be activated by acidification to yield chymosin with the same milk-clotting activity as the native enzyme (Marston et al., 1984). This finding suggests that recombinant prochymosin is similar to authentic prochymosin, but direct structural information is required to establish whether the zymogens are identical in conformation. We now describe fluorescence studies on authentic and recombinant prochymosin, and on pepsinogen, a closely related aspartic proteinase. Intrinsic fluorescence has been used to characterize the environment of tryptophan residues, and to study the sensitivity of the proteins to denaturation. Authentic and recombinant prochymosins were purified by the methods of Foltmann (1970) and Marston et al. (1 984) respectively. Porcine pepsinogen (Sigma) was also used. Fluorescence measurements were performed in a Perkin-Elmer MPF-3 fluorimeter thermostatically maintained at 25C, with excitation at 290 or 295nm, and excitation and emission bandwidths of 4-8 nm. The proteins were studied at approximately 10 pg/ml in Tris/HCl buffers or phosphate buffers. The intensity of fluorescence at the emission maximum was studied for all three proteins as a function of pH and denaturant concentration. Denatured proteins showed 50-60% reduced fluorescence. The denaturation fluorescence titrations indicate simple native (N) * denatured (D) transitions with no intermediate states detectable. At pH 7.5 the sensitivities of the three proteins to denaturation were identical; at pH 9.0 all three proteins were less stable and pepsinogen was clearly less stable than the prochymosins. Guanidine hydrochloride was effective as a denaturant at lower concentrations than urea (Table 1). Denaturation of prochymosins by guanidine hydrochloride was fully reversible, while urea denaturation was not. However, pepsinogen did recover native fluorescence intensity when the urea-denatured protein was returned to native conditions. Fluorescence was used to monitor the kinetics of unfolding when native proteins were transferred to denaturing conditions. Unfolding occurred on the time-scale 1-1000 s, and could be fully described by a single first-order rate constant ( k l ) . The values of k l increased with denaturant concentration, at a given pH, and with pH at a given denaturant concentration. Values of k l for authentic and recombinant prochymosin agreed to within ? 20% and both were greater than the values for pepsinogen by more than 10-fold. Studies on the kinetics of refolding suggest that there is a slow equilibration between alternative denatured states which have identical fluorescence properties. The fluorescence emission maximum for pepsinogen is at a shorter wavelength than that for prochymosins. Studies on fluorescence quenching by the uncharged polar dynamic quencher acrylamide suggest that the tryptophan residues of the three proteins are similar in accessibility and mobility as determined by the Stern-Volmer dynamic quenching constant k,. The fluorescence properties of pepsinogen and of authentic and recombinant prochymosin are summarized in Table 1. Whereas the prochymosins are closely similar in all the properties studied, the prochymosins are distinguishable from pepsinogen in (a) the wavelength of maximum emission of the native proteins, (b) the stability of the native state to denaturation by urea at pH 9.0, ( c ) the rate of unfolding in given conditions, ( d ) the reversibility of urea-denaturation.