Effect of Polyols and Salts on the Acid-Induced State of Human Placental Cystatin

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F. Rashid¹, S. Sharma¹, M. A. Baig², and B. Bano^1*

¹Department of Biochemistry, Faculty of Life Sciences, Aligarh Muslim University, Aligarh 202002, India; fax: (91) 5712-702-587; E-mail: fouzia_bc@yahoo.com; basiddiqui@hotmail.com

²Department of Biochemistry, Faculty of Science, Jamia Hamdard University, Hamdard Nagar, New Delhi 110056, India

^* To whom correspondence should be addressed.

Received June 16, 2005; Revision received January 25, 2006

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Polyols (glycerol and sorbitol) and salts (magnesium sulfate, sodium sulfate, and magnesium chloride) have been used to study the refolding of the acid-induced state of human placental cystatin (HPC), which is a low molecular weight (12,500 daltons) thiol proteinase inhibitor, in terms of CD spectroscopy, binding of hydrophobic dye 1-anilinonaphthalene-8-sulfonic acid (ANS), and intrinsic fluorescence measurements. The helical content of acid-denatured HPC increased with increase in glycerol concentration (0-80%). At 80% glycerol concentration, the secondary structural features observed in the far UV-CD region are similar to those of the native state (pH 6.0). The intrinsic fluorescence and near UV-CD studies showed that this 80% glycerol-induced state has a significant amount of tertiary structure with decreased ANS binding compared to the acid-denatured state. It was found that glycerol is more effective in stabilizing the acid-denatured state of HPC as compared to sorbitol. Among salts the stability effect was more for MgCl₂ (used up to concentration of 3 M) compared to MgSO₄ and Na₂SO₄ (used up to the concentration of 1.5 M due to restricted solubility of HPC at higher sulfate salt concentrations) as determined by CD studies and fluorescence measurements, which showed secondary and tertiary structural resemblance of this MgCl₂-induced state close to native state and showed overall spectral features in between the native state and the acid-denatured state. This MgCl₂ (3 M)-induced state showed decreased ANS fluorescence as compared to the acid-denatured state but more than that of the native state. The results taken together suggest that the acid-denatured state of HPC in the presence of 80% glycerol or 3 M MgCl₂ has a conformation in between that of the native state (pH 6.0) and the acid-induced state at pH 2.0.

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KEY WORDS: acid-induced state, human placental cystatin, circular dichroism, ANS, pH, protein folding

DOI: 10.11324/S0006297906060058

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