Title
Biophysical insight into structure-function relation of Allium sativum Protease Inhibitor by thermal, chemical and pH-induced modulation using comprehensive spectroscopic analysis
Author
Tooba Naz Shamsi, Romana Parveen, Huma Naz, Md. Anzarul Haque, Sadaf Fatima
Year
2017
Journal
International Journal of Biological Macromolecules
Abstract
In this study, we have analyzed the structural and functional changes in the nature of Allium sativum Protease Inhibitor (ASPI) on undergoing various denaturation with variable range of pH, temperature and urea (at pH 8.2). ASPI being anti-tryptic in nature has native molecular mass of ∼15 kDa. The conformational stability, functional parameters and their correlation were estimated under different conditions using circular dichroism, fluorescence and activity measurements. ASPI was found to fall in belongs to α+β protein. It demonstrated structural and functional stability in the pH range 5.0-12.0 and up to70 °C temperature. Further decrease in pH and increase in temperature induces unfolding followed by aggregation. Chemical induced denaturation was found to be cooperative and transitions were reversible and sigmoid. Tm (midpoint of denaturation), ΔCp (constant pressure heat capacity change) and ΔHm (van’t Hoff enthalpy change at Tm were calculated to be 41.25 ± 0.2 °C, 1.3 ± 0.07 kcal mol−1K−1 and 61 ± 2 kcal mol−1 respectively for thermally denatured ASPI earlier. The reversibility of the protein was confirmed for both thermally and chemically denatured ASPI. The results obtained from trypsin inhibitory activity assay and structural studies are found to be in a significant correlation and hence established structure-function relationship of ASPI.
Instrument
FP-6200
Keywords
Fluorescence, Protein structure, Chemical stability, Protein denaturation, Protein folding, Thermodynamics, Biochemistry