MIXTURES OF TRIFLUOROETHANOL OR HEXAFLUOROISOPROPANOL AND DIMETHYLFORMAMIDE ARE NOT OF GENERAL APPLICABILITY FOR PEPTIDE CONDENSATIONS CATALYZED BY TRYPSIN

Mixtures of a good hydrogen bond donor, 2,2,2-trifluoroethanol (TFE) o r 1,1,1,3,3,3-hexafluroisopropanol, and an acceptor, dimethylformamide (DMF) (1:1,v/v), containing 4% buffer have been described as adequate solvent systems for trypsin-catalyzed peptide fragment condensations [Mihara et al. (1993) Int. J. Pept. Protein Res. 41, 405]. Thus, we de cided to study the behaviour of trypsin in such solvent systems. We in vestigated whether this protease would efficiently catalyze condensati ons between fragments derived from an analogue of the gp-41 capsid pro tein of HIV virus or from cholecystokinin-22. None of the reactions ca rried out yielded the desired condensation products. However, when Fmo c-NLQNLDPSHR-OH and cholecystokinin-12 (H-ISDRDYMGWMDF-NH2) were used as substrates, the last had its R-D peptide bond hydrolyzed producing cholecystokinin-8. The proteolytic activity of this enzyme measured ag ainst a fluorogenic peptide derivative was 50 times lower in DMF/TFE c ontaining 5% of aqueous phase than in buffer. Steady-state fluorescenc e studies in DMF/TFE buffer were performed to examine the structure of this protease in these media. Steady-state spectra obtained with incr easing proportions of these two organic solvents in buffer showed that the emission intensities built up. Quenching studies with iodide reve aled that the I-o/I ratio (where I,and I are the fluorescence emission intensities in the absence and presence of quencher, respectively) ch anged from 1.2 in aqueous media to 2.2 in DMF/TFE (1:1, v/v) containin g 11% 0.2 M Tris-HCl buffer, pH 8.0, for 0.5 M iodide. The complete da ta indicated a higher exposure of tryptophan residues to the quencher in organic media, probably because of the partial unfolding of the enz yme. (C) Munksgaard 1997.