Effect of disaccharides on the stabilization of bovine trypsin against detergent and autolysis

Abstract

Osmolytes have been reported to stabilize biomolecules and even whole organisms against exposure to adverse environmental conditions. In this work, we report for the first time the use of some of these osmolytes, viz., the disaccharides trehalose and sucrose, in the stabilization of bovine trypsin against exposure to the anionic detergent sodium dodecyl sulfate and autolysis. Exposure of trypsin to SDS at a molar ratio of 1:45 led to decrease in trypsin activity by 61%. In the presence of 1 M sucrose and 1 M trehalose, the residual trypsin activity was found to increase to that of original enzyme activity. These two disaccharides were also found to slow down the rate of autolysis, resulting in residual activities of 80 and 88%, respectively, after incubation for 24 h. Active site titration showed retention of the fraction of active sites in the presence of trehalose. Fluorescence and CD spectroscopies were used to decipher the probable mechanism of this protective role of the disaccharides. Although complete resumption of secondary structure was not seen in the presence of the two disaccharides, the spectra of trypsin in the presence of stabilizers resembled the spectrum of native trypsin and were significantly different from the spectrum of detergent-denatured enzyme. Correlating the data obtained from spectroscopy with those obtained from activity assay, we propose that the retention of secondary structure of the enzyme is largely responsible for the retention of the functionally active form of trypsin.

Effect of Four Commonly Used Dissolution Media Surfactants on Pancreatin Proteolytic Activity

“…Porter and Peterson also observed this phenomenon, as the SDS-induced trypsin and chymotrypsin denaturation is slowed in presence of macromolecular substrates (34). There are also data in the literature that suggests that the addition of a sucrose, trehalose, or trypsin inhibitor to trypsin solutions may minimize the destabilizing effect of SDS and reduced the trypsin autoproteolysis (33,35).…”

“…It appears that the thermal treatment of the samples increased the unfolding rate of the proteinases that comprise the pancreatin. In comparison, Prasad et al reported that a 30-min incubation of trypsin at room temperature with various amounts of SDS (trypsin/SDS ratio from 1:10 to 1:80) reduced its initial activity by 61% (33).…”

Supercritical fluid assisted production of micrometric powders of the labile trypsin and chitosan/trypsin composite microparticles

“…8(a), the CD spectra (molar residue ellipticity (u) versus wavelength) of commercial trypsin in water exhibited a minimum around 208 nm and there was a hump around 220-222 nm wavelength for both spectra denoting the existence of a-helix, since bovine trypsin is a protein consisted of a-helix, b-sheet, b-turn and random coil (Ghosh, 2008). Although the general spectral shape remained, the ellipticity of processed trypsin at $208 nm and 210-220 nm band was reduced to nearly half the value of the native protein, indicating a structural perturbation of the above secondary structures (Prasad and Roy, 2010;Simon et al, 2001). Fig.…”

“…The maximum intensity of emission spectrum of commercial trypsin was observed at $340 nm. No red shift of the peak occurred, but the intensity of the spectra of processed trypsin decreased in comparison with the native curve, which meant that the microenvironment of the tryptophan and tyrosine residues was altered to some degree after SAA-HCM processing (Prasad and Roy, 2010). From the above discussion, the potential variations of trypsin molecular were implied, which might affect the biological activity of the enzyme.…”

The Electrostatic Interactions Between Nano-TiO2 and Trypsin Inhibit the Enzyme Activity and Change the Secondary Structure of Trypsin

“…The two domains of trypsin comprised mainly H-bonded crossed β-sheet networks. One of the sheets positioned the catalytic Ser correctly in the active site, whereas the second sheet formed a part of the substrate recognition cleft [27]. It could be inferred that β-sheet of trypsin bound directly to the TiO 2 nanoparticles, and the press and pull strength twisted the sheets, which further resulted in the inactivation of trypsin.…”