Effect of polyols and salts on the acid-induced state of human placental cystatin.
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 MgCl(2) (used up to concentration of 3 M) compared to MgSO(4) and Na(2)SO(4) (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 MgCl(2)-induced state close to native state and showed overall spectral features in between the native state and the acid-denatured state. This MgCl(2) (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 MgCl(2) has a conformation in between that of the native state (pH 6.0) and the acid-induced state at pH 2.0.
Rashid
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Department of Biochemistry, Faculty of Life Sciences, Aligarh Muslim University, Aligarh, 202002, India.
Sharma
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Baig
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MA
Bano
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B
D002942:Circular Dichroism; D015891:Cystatins / Q000737:chemistry* / Q000378:metabolism; D005454:Fluorescence Polarization; D005990:Glycerol / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D015636:Magnesium Chloride / Q000494:pharmacology; D008278:Magnesium Sulfate / Q000494:pharmacology; D011108:Polymers / Q000494:pharmacology*; D011257:Pregnancy Proteins / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation / Q000187:drug effects*; D011489:Protein Denaturation / Q000187:drug effects; D012492:Salts / Q000494:pharmacology*; D013012:Sorbitol / Q000494:pharmacology; D013431:Sulfates / Q000494:pharmacology
D015891:Cystatins; D011108:Polymers; D011257:Pregnancy Proteins; D012492:Salts; D013431:Sulfates; C024617:polyol; D015636:Magnesium Chloride; C012036:sodium sulfate; D013012:Sorbitol; D008278:Magnesium Sulfate; D005990:Glycerol