Changes of fluorescence lifetime and rotational correlation time of bovine serum albumin labeled with 1-dimethylaminonaphthalene-5-sulfonyl chloride in guanidine and thermal denaturations |
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Authors: | Kunio Takeda Isao Yoshida and Kazuo Yamamoto |
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Institution: | (1) Department of Applied Chemistry, Okayama University of Science, 1-1 Ridai-cho, 700 Okayama, Japan |
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Abstract: | The nanosecond fluorescence depolarization method was applied to measure the fluorescence lifetime ( ) and the rotational correlation time ( ) of bovine serum albumin (BSA) labeled with 1-dimethylaminonaphthalene-5-sulfonyl chloride (dansyl-Cl). Changes of and of dansyl BSA in the guanidine denaturation and in the thermal denaturation were examined. In parallel, the secondary structural change of dansyl BSA was followed by circular dichroism measurements. The magnitude of was almost unchanged between 1 and 2 M guanidine, where the secondary structure of the protein was predominantly disrupted; whereas that of began to increase before the disruption of secondary structure in the guanidine denaturation. In the thermal denaturation, in contrast, changes of both and occurred in a temperature range where the secondary structure was predominantly disrupted. The volume of equivalent sphere (V
e
) and the axial ratio ( ) for the BSA were 3.6–3.8×10–19 cm3 and 3.6 at 2M guanidine as against 2.1×10–19 cm3 and 2.2 in the absence of guanidine (25°C), respectively. The magnitudes ofV
e
and were 4.9×10–19 cm3 and 4.5 at 65°C, respectively. Although the secondary structural change of dansyl BSA was irreversible in the thermal denaturation,V
e
and were reversible. |
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Keywords: | Bovine serum albumin fluorescence lifetime fluorescence anisotropy denaturation |
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