MIXTURES OF TRIFLUOROETHANOL OR HEXAFLUOROISOPROPANOL AND DIMETHYLFORMAMIDE ARE NOT OF GENERAL APPLICABILITY FOR PEPTIDE CONDENSATIONS CATALYZED BY TRYPSIN
Mp. Bemquerer et al., MIXTURES OF TRIFLUOROETHANOL OR HEXAFLUOROISOPROPANOL AND DIMETHYLFORMAMIDE ARE NOT OF GENERAL APPLICABILITY FOR PEPTIDE CONDENSATIONS CATALYZED BY TRYPSIN, The journal of peptide research, 51(1), 1998, pp. 29-37
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.