Protein stability function relations: beta-lactoglobulin-A sulphydryl group reactivity and its relationship to protein unfolding stability.
The effect of protein stability on the reactivity of the free sulphydryl (SH) group in beta-lactoglobulin-A (beta-LgA) provides a model for the study of protein stability-function relations (PSFR). The free energy change for protein unfolding (delta G(o)) and SH group exposure (delta GSH) were determined from (i) the urea unfolding curve for beta-LgA and (ii) the kinetics of beta-LgA SH/disulphide exchange with 2-pyridine disulphide (2-PDS) in 0-8 M urea (pH 3). Protein unfolding profiles determined from extrinsic fluorescence and SH-group reactivity measurements were not coincident. beta-LgA formed a stable intermediate (X) state in the presence of 4 M urea with delta G(o) = 20 (+/- 0.03) kJ/mol. From the low rate of SH/disulphide exchange in 4 M urea, the SH-group within beta-LgA was efficiently masked within the X-state. SH reactivity increased after beta-LgA was unfolded in 6-8 M urea with, delta GSH = 43(+/- 6.4) kJ/mol. Such results are discussed in terms of possible interrelationships between protein unfolding stability and SH reactivity in beta-LgA.
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Procter Department of Food Science, School of Physical Sciences, University of Leeds, UK. R.K.Owusu@Leeds.ac.uk
D004355:Drug Stability; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D007782:Lactoglobulins / Q000737:chemistry*; D008956:Models, Chemical; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013438:Sulfhydryl Compounds / Q000737:chemistry; D013816:Thermodynamics
D007782:Lactoglobulins; D013438:Sulfhydryl Compounds