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Journal of Biochemistry Advance Access published online on February 16, 2007
Journal of Biochemistry, doi:10.1093/jb/mvm057
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© 2007 The Japanese Biochemical Society

Different molten globule like folding intermediates of hen egg white lysozyme induced by high pH and tertiary butanol

Mahrukh Hameed1, Basir Ahmad2, Khalid Majid Fazili1, Khurshid Andrabi1 and Rizwan Hasan Khan2,*

1Department of Biotechnology, University of Kashmir, Hazratbal, Srinagar -190006, India
2Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh – 202002, India.

*Corresponding author : Rizwan H. Khan, Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh - 202 002, INDIA. E-mail: rizwanhkhan{at}hotmail.com, rizwanhkhan1{at}yahoo.com Tel: + 91-571-2720388 Fax: + 91-571-2721776

Received December 9, 2006; Accepted February 7, 2007


  Abstract

We have provided evidence that hen egg white lysozyme (HEWL) existed in {alpha} helical and ß structure dominated MG states at high pH and in the presence of tertiary butanol respectively. Circular dichroism (CD), intrinsic fluorescence, ANS binding and acrylamide induced fluorescence quenching techniques have been used to investigate alkali induced unfolding of HEWL and the effect of tertiary butanol on the alkaline induced state. At pH 12.75 HEWL existed as molten globule like intermediate. The observed MG like intermediate was characterized by (i) retention of 77% of the native secondary structure (ii) enhanced binding of ANS (~ 5 times) compared to native and completely unfolded state (iii) loss of the tertiary structure as indicated by the tertiary structural probes (near UV CD and Intrinsic fluorescence) and (iv) acrylamide quenching studies showed that MG state has compactness intermediate between native and completely unfolded states. Moreover structural properties of the protein at PI and denatured states have also been described. We have also shown that in the presence of 45% t-butanol HEWL at pH 7.0 and pH 11.0 (pI 11.0) existed in helical structure without much affecting tertiary structure. Interestingly, MG state of HEWL at pH 12.7 transformed into another molten globule state (MG2) at 20% t-butanol (v/v), in which secondary structure is mainly ß sheets. On further increasing the t-butanol concentration {alpha} helix was found to reform. We have proposed that formation of both {alpha} helical and ß sheet dominated intermediate may be possible in the folding pathway of {alpha} + ß protein.

Key Words: Alkali induced unfolding, Circular dichroism, Fluorescence quenching, Lysozyme, Molten globule


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