TY - JOUR
T1 - Effect of pH on thermal- and chemical-induced denaturation of GFP
AU - Alkaabi, Klaithem M.
AU - Yafea, Abeer
AU - Ashraf, S. Salman
N1 - Funding Information:
The authors would like to thank the UAE Research Affairs department for financial supports for K. M. A. and A. Y. (Grant no. 03-03-2-11/04 to SSA.)
PY - 2005/8
Y1 - 2005/8
N2 - Green fluorescent protein (GFP) is an unusually stable autofluorescent protein that is increasingly being exploited for many applications. In this report, we have used fluorescence spectroscopy to study the effect of pH on the denaturation of GFP with sodium dodecyl sulfate (SDS), urea, and heat. Surprisingly, SDS (up to 0.5%) did not have any significant effect on the fluorescence of GFP at pH 7.5 or 8.5 buffers; however, at pH 6.5, the protein lost all fluorescence within 1 min of incubation. Similarly, incubation of GFP with 8 M urea at 50°C resulted in time dependent denaturation of GFP, but only in pH 6.5 buffer. At higher pH values (pH 7.5 and pH 8.5), the GFP was quite stable in 8 M urea at 50°C, showing only a slight decrease in fluorescence. Heat denaturation of GFP was found to be pH dependent as well, with the denaturation being fastest at pH 6.5 as compared to pH 7.5 or pH 8.5. Like the denaturation studies, renaturation of heat-denatured GFP was most efficient at pH 8.5, followed by pH 7.5, and then pH 6.5. These results suggests that GFP undergoes a structural/stability shift between pH 6.5 and pH 7.5, with the GFP structure at pH 6.5 being very sensitive to denaturation by SDS, urea, and heat.
AB - Green fluorescent protein (GFP) is an unusually stable autofluorescent protein that is increasingly being exploited for many applications. In this report, we have used fluorescence spectroscopy to study the effect of pH on the denaturation of GFP with sodium dodecyl sulfate (SDS), urea, and heat. Surprisingly, SDS (up to 0.5%) did not have any significant effect on the fluorescence of GFP at pH 7.5 or 8.5 buffers; however, at pH 6.5, the protein lost all fluorescence within 1 min of incubation. Similarly, incubation of GFP with 8 M urea at 50°C resulted in time dependent denaturation of GFP, but only in pH 6.5 buffer. At higher pH values (pH 7.5 and pH 8.5), the GFP was quite stable in 8 M urea at 50°C, showing only a slight decrease in fluorescence. Heat denaturation of GFP was found to be pH dependent as well, with the denaturation being fastest at pH 6.5 as compared to pH 7.5 or pH 8.5. Like the denaturation studies, renaturation of heat-denatured GFP was most efficient at pH 8.5, followed by pH 7.5, and then pH 6.5. These results suggests that GFP undergoes a structural/stability shift between pH 6.5 and pH 7.5, with the GFP structure at pH 6.5 being very sensitive to denaturation by SDS, urea, and heat.
KW - E. coli
KW - GFP
KW - Renaturation
KW - SDS
KW - Thermal denaturation
KW - Urea
UR - http://www.scopus.com/inward/record.url?scp=24044492592&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=24044492592&partnerID=8YFLogxK
U2 - 10.1385/ABAB:126:2:149
DO - 10.1385/ABAB:126:2:149
M3 - Article
C2 - 16118468
AN - SCOPUS:24044492592
SN - 0273-2289
VL - 126
SP - 149
EP - 156
JO - Applied Biochemistry and Biotechnology
JF - Applied Biochemistry and Biotechnology
IS - 2
ER -