Md. Anzarul Haque1, Shah Ubaid-ullah1, Sobia Zaidi1, Md. Imtiayaz Hassan1, JK Batra2, Faizan Ahmad1
1Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, Jamia Nagar, New Delhi – 110025, India.
2Immuno-chemistry Lab, National Institute of Immunology, New Delhi -1100 67, India.
ABSTRACT
Cytochrome c is a member of an extended family of heme-proteins involved in electron transfer functions in the mitochondrial respiratory chain, bacterial photosynthesis and apoptosis. More than 270 sequences of cytochromes c have been reported. Multiple sequence alignment suggests that the Yeast iso-1-cytochrome c has five extra residues at the N-terminal of the polypeptide chain. In order to investigate the role of these amino acid residues in the stability of the protein, we constructed variants of the protein deleting each amino acid residue. These variants, ∆ (-5/-1), ∆ (-5/-2), ∆ (-5/-3), ∆ (-5/-4) and ∆ (-5/-5) were expressed in BL21 and subsequently purified. Heat-induced denaturation of these variants and wild type protein in the native buffer were measured by monitoring changes in absorbance at 405nm (∆ε405) and CD at 222nm ([θ]222 ) in the temperature range 20-80°C. The data were fitted to the two-state denaturation model and values of Tm (midpoint of denaturation) and ∆Hm (change in enthalpy at Tm ) were determined. We observed that the Tm of all the variants except ∆ (-5/-4) decreased considerably in comparison to the wild type protein. Read more…
