Influence of extrinsic factors on guanidine hydrochloride denaturation of Bacillus licheniformis α-amylase / Salanee Kandandapani
The influence of buffer composition on the conformational stability of native and calcium-depleted Bacillus licheniformis α-amylase (BLA) was investigated against guanidine hydrochloride (GdnHCl) denaturation using circular dichroism, fluorescence and UV-difference spectroscopy. Buffers used in t...
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Format: | Thesis |
Published: |
2016
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Online Access: | http://studentsrepo.um.edu.my/9269/1/Salanee_Kandanpani.pdf http://studentsrepo.um.edu.my/9269/6/THESIS_FINAL_2016_(November)_1st_edit_copy.pdf http://studentsrepo.um.edu.my/9269/ |
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Summary: | The influence of buffer composition on the conformational stability of native
and calcium-depleted Bacillus licheniformis α-amylase (BLA) was investigated against
guanidine hydrochloride (GdnHCl) denaturation using circular dichroism, fluorescence
and UV-difference spectroscopy. Buffers used in these experiments were:
0.05 M sodium phosphate buffer, pH 7.5, 0.15 M Tris-HCl buffer, pH 7.5,
0.15 M HEPES buffer, pH 7.5 and 0.15 M MOPS buffer, pH 7.5. Differential effects of
buffer composition on GdnHCl denaturation of BLA were evident from the magnitude
of these spectral signals, which followed the order: sodium phosphate > Tris-HCl >
HEPES > MOPS. These effects became more pronounced when calcium-depleted BLA
was used in the incubation mixture as revealed by a lower relative mean residue
ellipticity, lower relative fluorescence intensity, and higher change in emission
maximum. Depletion of calcium from BLA suggested a decrease in the protein
conformational stability. Gel chromatographic analyses of native, 3 M GdnHCldenatured
and 6 M GdnHCl-denatured BLAs were made in different runs on Sephacryl
S-200 HR column (1.0×30 cm), equilibrated with these buffers. The results obtained
clearly suggested formation of similar denatured states and aggregated forms of BLA in
3 M and 6 M GdnHCl in the presence of these buffers. However, quantitative
differences in BLA aggregation were noticed in these buffers in the presence of 6 M
GdnHCl. In view of the above, spectral results on BLA stability against GdnHCl
obtained with different probes (MRE, fluorescence intensity and emission maximum) in
different buffers should be treated with caution. |
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