Fluorescence study of guanidine hydrochloride denaturation of thymidylate synthetase☆

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Abstract

The denaturation of thymidylate synthetase by guanidine hydrochloride has been studied using both the intrinsic fluorescence of the protein, and the polarization of the 1-dimethyl aminonaphthalene 5-sulfonyl conjugate of the protein. The polarization of the conjugate shows two transitions. The first transition, complete by 2.3 M guanidine, involves swelling or elongation of the protein; the second, complete by 5.5 M guanidine, is associated with unfolding of the protein. The Stokes' shift of the intrinsic protein fluorescence reflects a transition which is complete by 5.0 M guanidine hydrochloride.

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This research was supported by Grant CH-25 from the American Cancer Society and by USPHS Grant CH-13148. The authors thank Dr. Stephen C. Harvey for assistance in performing this investigation.

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