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Structural stability and refolding of the soybean Kunitz trypsin inhibitor

Robin Roychaudhuri, University of Nebraska - Lincoln

Abstract

Soybean Kunitz trypsin inhibitor (SKTI) is a minor allergen found in foods containing soy proteins. Apart from being a food allergen, it has also played a key role in elucidating the mechanism of protease-protease inhibitor interactions that has helped in our better understanding of the action of proteases. SKTI has two well-conserved disulfide bridges that play an important part in its structure. Proteolysis studies on reduced SKTI were performed with different reducing agents and its stability assessed in time course experiments. ^ Thermal denaturation studies were done on SKTI due to its refractoriness to conventional chemical denaturants and also since foods generally undergo heat treatment and processing. During the process of thermal denauration at both controlled and different rates, the different conformations of SKTI were probed using proteases like chymotrypsin and pepsin. This was done to assess their similarity with the native conformation that is protease resistant. Conformational transitions during thermal denaturation were monitored using 8-anilino-1-naphthalene sulfonic acid (ANS) fluorescence, intrinsic tryptophan fluorescence, CD and UV absorbance spectroscopy. Structure-function relation studies were also done on SKTI during thermal denaturation of native and reduced inhibitor. ^ Acid denaturation studies were done on SKTI to mimic the gastrointestinal environment that these food allergens are routinely exposed to. Structural transitions in SKTI were monitored in Simulated Gastric Fluid and in HCl-water mixtures using far and near UV CD and ANS fluorescence spectroscopy. ^ The research presented here represents a protein structural stability study of an allergen like SKTI. Hence in the context of protein structure and folding, its allergenic potential is discussed. ^

Subject Area

Agriculture, Food Science and Technology|Chemistry, Biochemistry|Biophysics, General|Health Sciences, Immunology

Recommended Citation

Roychaudhuri, Robin, "Structural stability and refolding of the soybean Kunitz trypsin inhibitor" (2003). ETD collection for University of Nebraska - Lincoln. AAI3078610.
http://digitalcommons.unl.edu/dissertations/AAI3078610

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