Characterization of molten globule state of fetuin at low pH.
Effect of pH over a range of 0.8-10 on bovine serum fetuin (BSF) was observed by far and near-UV circular dichroism (CD) spectroscopy, intrinsic tryptophan fluorescence and ANS fluorescence measurements. It has been reported earlier by our group that a molten globule (MG) state exists in alpha-chymotrypsinogen [Biochim. Biophys Acta 1481 (2000) 229] and stem bromelain [Eur. J. Biochem. 269 (2002) 47] at low pH. In this paper we have shown the presence of an MG form of fetuin at low pH. The far-UV CD spectra showed the regain of secondary structure at pH 1.8 as compared to the complete loss of secondary structure in presence of 6 M GnHCl. Near-UV CD spectra showed disruption of tertiary structure at pH 1.8. Tryptophan fluorescence studies indicated that there is only a slight red shift in the wavelength emission maxima (lambdamax) of fetuin at low pH as compared to a significantly red-shifted spectrum of the completely unfolded state in 6 M GnHCl, indicating that the tryptophan environment of fetuin at low pH resembles more the native form. ANS binding experiments also showed an enhancement in ANS binding with decrease in pH up to 1.8. ANS binding was absent at pH 7 and in the presence of 6 M GnHCl. Fluorescence quenching experiments were also performed with acrylamide, cesium chloride and potassium iodide. The quenching of tryptophan fluorescence by the three different quenchers indicates that low pH induces a conformational change in protein, making the tryptophan residue less accessible to solvent. This suggests that a more compact structure exists at low pH. The results, being in accordance with far-UV CD and fluorescence studies, imply the presence of MG state of fetuin at low pH. As studied by fluorescence spectroscopy, denaturation of fetuin at low pH was found to be reversible.
Naseem
Farah
F
Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh 202002, India.
Khan
Rizwan Hasan
RH
Haq
Soghra Khatun
SK
Naeem
Aabgeena
A
D000818:Animals; D002417:Cattle; D002942:Circular Dichroism; D006863:Hydrogen-Ion Concentration; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence; D013696:Temperature; D014364:Tryptophan / Q000737:chemistry; D000509:alpha-Fetoproteins / Q000737:chemistry*
D000509:alpha-Fetoproteins; D014364:Tryptophan