Time Evolution of Exposed Hydrophobicity of Water-Soluble Proteins During their Depolymerisation by Endo-Proteases

Abstract

During the depolymerisation of a water-soluble protein by an endo-protease, the exposed hydrophobicity of the substrate, that is the hydrophobicity that is accessible to hydrophobic probes, changes with the progress of the reaction. This work describes the depolymerisation of bovine serum albumin, α-casein and β-lactoglobulin using the proteases Alcalase, Flavourzyme, α-chymotrypsin, mercuripapain and trypsin. Time evolution of substrate hydrophobicity was monitored by a flow-injection analysis (FIA) system with fluorescence detection and an aqueous eluant containing p-toluidinylnaphthalene-6-sulfonate (2,6-TNS) as the fluorescent probe. In all cases, the time evolution of the substrate hydrophobicity was fitted using a derived mathematical function containing two adjustable rate constants and two constant parameters. This methodology allowed the determination of protease activities, as well as online monitoring of the depolymerisation process, when using water-soluble proteins as substrate.

Keywords

depolymerisation enzymes proteins kinetics hydrophobicity

Get full access to this article

View all access options for this article.

References

Adler-Nissen J. (1979). Determination of the degree of hydrolysis of food protein hydrolysates by trinitrobenzenesulfonic acid . Journal of Agriculture and Food Chemistry 27: 1256-1262 .

Batlle N. , Carbonell J.V. and Sendra J.M. (2000). Determination of the depolymerization kinetics of amylose, amylopectin and soluble starch by Aspergillus oryzae α-amylase using a fluorimetric 2-p-toluidinylnaphthalene-6-sulfonate/flow-injection analysis system . Biotechnology and Bioengineering 70: 544-552 .

Green F.C. and Kasarda D.D. (1971). Apolar interactions of α-gliadin: binding of 2-p-toluidinylnaphthalene-6-sulfonate . Cereal Chemistry 48: 601-609 .

McClure W.O. and Edelman G.M. (1966). Fluorescent probes for conformational states of proteins. I. Mechanism of fluorescence of 2-p-toluidinylnaphthalene-6-sulfonate, a hydrophobic probe . Biochemistry 5: 1908-1919 .

Sendra J.M. and Carbonell J.V. (1998). A theoretical equation describing the time evolution of the concentration of a selected range of substrate molecular weights in depolymerization processes mediated by single-attack mechanism endo-enzymes . Biotechnology and Bioengineering 57: 387-393 .

Sentandreu E. , Carbonell J.V. and Sendra J.M. (2002). Monitoring of chemical and enzymatic hydrolysis of water-soluble proteins using flow-injection analysis with fluorescence detection and an aqueous eluant containing 2-p-toluidinylnaphthalene-6-sulfonate as the fluorescent probe . Biotechnology and Bioengineering 78: 828-832 .

Silvestre M.P.C. , Hamon M. and Yvon M. (1994). Analysis of protein hydrolysates. I. Use of poly(2-hydroxyethylaspartamide)-silica column in size exclusion chromatography for the fractionation of casein hydrolysates . Journal of Agriculture and Food Chemistry 42: 2778-2782 .

Strickland D.K. , Steiner J.P. , Feldman S.R. and Pizzo S.V. (1984). Fluorescent probes as a measure of conformational alterations induced by nucleophilic modification and proteolysis of bovine alpha 2-macroglobulin . Biochemistry 23: 6679-6685 .