title: Loosening of globular structure under alkaline pH affects accessibility of β-lactoglobulin to tyrosinase-induced oxidation and subsequent cross-linking.
abstract: Globular proteins such as β-lactoglobulin (BLG) are poorly accessible to enzymes. We have studied susceptibility of BLG to oxidation by Trichoderma reesei (TrTyr) and Agaricus bisporus (AbTyr) tyrosinases and subsequent intermolecular cross-linking with respect to pH-induced structural changes. We evaluated pH-induced structural changes in BLG using circular dichroism, tryptophan fluorescence and small angle X-ray scattering (SAXS) measurements, where after these results were correlated with the analysis of cross-linking by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Oxygen consumption measurement and changes in radii of gyration determined by SAXS during the enzyme-induced oxidation at the respective reaction conditions were also followed. Intermolecular cross-linking of BLG by TrTyr was found at pH 9 but not at pH 7.5. AbTyr was unable to catalyze cross-linking at pH 7.5 or pH 9. Increased accessibility and cross-linking by TrTyr was addressed to loosening of the three dimensional structure of the protein, increased flexibility of the backbone as well as partial hydrolysis. In addition to basic research of the effect of protein folding on enzymatic cross-linking the research results have significance on the exploitation of TrTyr at alkaline conditions.
authors: VTT Technical Research Centre of Finland, P.O Box 1000, FI-02044 VTT, Finland. riitta.partanen@vtt.fi
authors: Riitta
authors: R
authors: Partanen
authors: Mika
authors: M
authors: Torkkeli
authors: Maarit
authors: M
authors: Hellman
authors: Perttu
authors: P
authors: Permi
authors: Ritva
authors: R
authors: Serimaa
authors: Johanna
authors: J
authors: Buchert
authors: Maija-Liisa
authors: ML
authors: Mattinen
mesh_terms: D000818:Animals; D002417:Cattle; D002942:Circular Dichroism; D003432:Cross-Linking Reagents; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007782:Lactoglobulins / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D014442:Monophenol Monooxygenase / Q000378:metabolism; D010084:Oxidation-Reduction; D011487:Protein Conformation; D053838:Scattering, Small Angle; D013050:Spectrometry, Fluorescence; D014961:X-Ray Diffraction
chemical_list: D003432:Cross-Linking Reagents; D007782:Lactoglobulins; D014442:Monophenol Monooxygenase