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Journal of Biochemistry Advance Access originally published online on May 15, 2007
Journal of Biochemistry 2007 142(1):65-72; doi:10.1093/jb/mvm108
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© 2007 The Japanese Biochemical Society.

Effect of Polyethylene Glycols on the Function and Structure of Thiol Proteases

Sadaf Fatima and Rizwan Hasan Khan*

Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh– 202 002, India

*To whom correspondence should be addressed. Tel: +91-571-2720388, Fax: + 91-571-2721776, E-mail: rizwanhkhan{at}hotmail.com or rizwanhkhan1{at}yahoo.com

Received December 19, 2006; Accepted April 21, 2007


  Abstract

Thiol proteases are industrially significant proteins with catalytic efficiency. The effect of low, medium and high molecular-weight poly (ethylene glycol) (PEG- 400, 6000 and 20000) on the stability of thiol proteases (papain, bromelain and chymopapain) has been studied by activity measurements using synthetic substrate. Structural studies performed on papain by far UV circular dichroism spectroscopic measurements indicate that there is loss in secondary structure of the protein in presence of increasing concentration of PEGs. Intrinsic fluorescence measurements lead us to conclude that tryptophan residues of protein encounter non-polar microenvironment in presence of PEG solvent while acrylamide quenching shows greater accessibility of tryptophan residues of papain in presence of PEGs. Extrinsic fluorescence measurements lead us to conclude that PEGs bind to the hydrophobic sites on the protein and thus destabilize it. Thermal denaturation studies show that melting temperature of papain is decreased in presence of PEGs. Possible mechanism of destabilization is discussed next. The results imply that caution must be exercised in the use of PEGs with thiol proteases or hydrophobic proteins in general, for different industrial applications, even at room temperature.

Key Words: Poly(ethylene glycol), protein stability, synthetic substrate, thiol proteases

Abbreviations: ANS, 8-anilino-1-naphthalene-sulphonic acid; DMSO, dimethylsulphoxide; EDTA, ethylene diamine tetra-acetic acid; GdnHCl, guanidine hydrochloride; MRE, mean residue ellipticity; NATA, N-acetyl tryptophanamide; PEG, poly(ethylene glycol); Tm, mid point of transition; w/v, weight/volume ratio


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