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Journal of Biochemistry Advance Access originally published online on March 15, 2008
Journal of Biochemistry 2008 144(1):33-38; doi:10.1093/jb/mvn036
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© 2008 The Japanese Biochemical Society

Bromophenol Blue Binding as a Probe to Study Urea and Guanidine Hydrochloride Denaturation of Bovine Serum Albumin

Adyani Azizah Abd. Halim, Habsah Abdul Kadir and Saad Tayyab*

Biomolecular Research Group, Biochemistry Programme, Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia

*To whom correspondence should be addressed. Tel: +60-3-7967-7118, Fax: +60-3-7967-4178, E-mail: saadtayyab2004{at}yahoo.com

Received November 9, 2007; Accepted March 7, 2008


  Abstract

Urea and guanidine hydrochloride (GdnHCl) denaturation of bovine serum albumin (BSA) were investigated using bromophenol blue (BPB) binding as a probe. Addition of BPB to BSA produced an absorption difference spectrum in the wavelength range, 525–675 nm with a minimum at 587 nm and a maximum at 619 nm. The magnitude of absorption difference ({Delta}Abs.) at 619 nm decreased on increasing urea/GdnHCl concentration and followed the denaturation curve. The denaturation was found to be a two-state, single-step transition. The transitions started at 1.75 and 0.875 M and completed at 6.5 and 3.25 M with the mid point occurring around 4.0 and 1.5 M urea and GdnHCl concentrations, respectively. The value of free energy of stabilization, Formula as determined from urea and GdnHCl denaturation curves was found to be 4041 and 4602 cal/mol, respectively. Taken together, these results suggest that BPB binding can be used as a probe to study urea and GdnHCl denaturation of BSA.

Key Words: bovine serum albumin, bromophenol blue, denaturation, guanidine hydrochloride, urea

Abbreviations: {Delta}Abs., absorbance difference; ANS, 1-anilino-naphthalene-8-sulfonate; BSA, bovine serum albumin; BPB, bromophenol blue; GdnHCl, guanidine hydrochloride; HSA, human serum albumin; UV, ultraviolet


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