Raman Studies on Native, Reduced, and Modified Basic Pancreatic Trypsin Inhibitor

Abstract

The method of Raman scattering was applied to determine the effect of chemical modification on the conformation of the basic pancreatic trypsin inhibitor in aqueous solution. Cleavage of one of the three disulfide bonds yields no spectral changes except, of course, the manifestation of the reduction, i.e. a lower intensity of the disulfide band and the appearance of a line due to the newly formed sulfhydryl groups. S-alkylation, however, produces several changes in the spectra as compared to the native inhibitor. In the case of the carboxymethyl derivative the amide I11 band, shifted to lower frequency, is particularly significant. This observation suggests that the inactivity towards trypsin of the reduced carboxymethyl inhibitor is the result of structural changes near the reactive centre of the molecule.The basic pancreatic trypsin inhibitor (trypsin kallikrein inhibitor) is one of the smallest globular proteins known today. At neutral pH values it forms an unusually stable complex with trypsin. It shows no loss of activity after exposure to very acidic solutions (pH 0.5) or high temperatures (95 "C) [1-31. The amino acid sequence [4][5][6] and the threedimensional structure in single crystals [7,8] are known. The polypeptide chain of the molecule is fixed by three disulfide bridges. The Cys-14-Cys-38 disulfide bond close to the reactive centre can be reduced selectively [9].In recent years Raman spectroscopy has proved to be a valuable method for the investigations of protein conformations in solution. Lately, we have compared the Raman spectra of the basic pancreatic inhibitor in aqueous solution and in single crystal [lo] and found evidence for a different conformation of the inhibitor in both states. In this investigation the Raman technique was used to study the conformationa1 properties of native and chemically modified basic pancreatic inhibitor at ambient and higher temperatures. A preliminary report of this work has been presented [I 11. MATERIALS AND METHODSThe trypsin inhibitor (Trasylol") was a gift of Verfahrensentwicklung Biochemie, Bayer Leverkusen. The freeze-dried samples (Lot GOS 674/52 and GOS 365/52) where homogeneous as judged from polyacrylamide electrophoresis. The amino acid analyses were in excellent agreement to those found previously.Reduced trypsin inhibitor, with the disulfide bonds Cys-14 -Cys-38 selectively cleaved, was prepared according to the method of Kress and Laskowski [9]. Completeness of reduction was checked by measuring the amount of -SH groups with 5,5'-dithio-bis(2-nitrobenzoic acid) [12]. After acidification to pH 2.0 in order to destroy the excess of sodium borohydride, part of the solution (43 mg protein/ml) was used to record the spectra ofthe reduced inhibitor. No reoxidation took place as was confirmed by determination of the number of sulfhydryl groups after the experiments. The other portion was reacted with iodoacetic acid [9] and with iodoacetamide [13], respectively, according to the procedures of Kress and Laskowski. The S-alkylated derivatives, reduced c...

Complete tyrosine assignments in the high-field proton nuclear magnetic resonance spectrum of bovine pancreatic trypsin inhibitor selectively reduced and carboxamidomethylated at cystine 14-38

“…These results are consistent with earlier laser Raman (Brunner et al, 1974) and CD/ORD (Vincent et al, 1971) spectroscopic studies showing few differences at physiological conditions when comparing RCAM-unnitrated and native BPTI. The aggregate alkaline isomerization curves measured as A[m]230nm between pH 7.5 and 11.5 were exactly superimposable for RCAM-unnitrated and native BPTI at 25 °C in 0.1 M NaCl.…”

Raman spectroscopy of proteins: a review

“…Moreover, this protein is highly stable and very resistant toward denaturation. [117] The first comprehensive studies on TIP were carried out by Brunner et al [118] with the use of 488 nm laser, but also the FTRS analysis of TIP thermal stability and conformation was published. [117] Our results are in accordance with literature data.…”

Selective characterization of proteins on nanoscale concave surfaces

“…Using the static scan mode at amide III bands in Figure 3B, peak doublets were observed at 1250 cm -1 and 1300 cm -1 . The peaks at 1300 cm -1 were assigned to α-helices, and the broad peaks around 1250 cm -1 was assigned to the overlap of β-sheets (~1243 cm -1 ) and random coils (~1265 cm -1 ) [41]. The α-helix content of each sample was quantified, and an estimation of α-helix content is given in Figure 3C.…”