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) or 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 decided to study the behaviour of trypsin in such solvent systems. We investigated whether this protease would efficiently catalyze condensations between fragments derived from an analogue of the gp-41 capsid protein of HIV virus or from cholecystokinin-22. None of the reactions carried out yielded the desired condensation products. However, when Fmoc-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 against a fluorogenic peptide derivative was 50 times lower in DMF/TFE containing 5% of aqueous phase than in buffer. Steady-state fluorescence studies in DMF/TFE buffer were performed to examine the structure of this protease in these media. Steady-state spectra obtained with increasing proportions of these two organic solvents in buffer showed that the emission intensities built up. Quenching studies with iodide revealed that the Io/I ratio (where Io and I are the fluorescence emission intensities in the absence and presence of quencher, respectively) changed from 1.2 in aqueous media to 2.2 in DMF/TFE (1:1, v/v) containing 11% 0.2 M Tris-HCl buffer, pH 8.0, for 0.5 M iodide. The complete data indicated a higher exposure of tryptophan residues to the quencher in organic media, probably because of the partial unfolding of the enzyme.
Bemquerer
M P
MP
Department of Biochemistry, University of São Paulo, Brazil.
Liria
C W
CW
Kitagawa
K
K
Miranda
M T
MT
Tominaga
M
M
D000595:Amino Acid Sequence; D002384:Catalysis; D004126:Dimethylformamide / Q000737:chemistry*; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D020005:Propanols / Q000737:chemistry*; D014270:Trifluoroethanol / Q000737:chemistry*; D014357:Trypsin / Q000378:metabolism*; D006845:Hydrocarbons, Fluorinated
D004126:Dimethylformamide; D010446:Peptide Fragments; D020005:Propanols; D014270:Trifluoroethanol; D014357:Trypsin; C001337:hexafluoroisopropanol; D006845:Hydrocarbons, Fluorinated