Preparation of Spin-Labeled Bacterial Ribonuclease from Bacillus intermedius 7P for EPR Studies of Protein Dynamics

L. O. Yagodina^1*, A. E. Altshuler¹, and Y. V. Abreimova²

¹Kazan Institute of Biochemistry and Biophysics, Kazan Research Center, Russian Academy of Sciences, ul. Lobachevskogo 2/31, Kazan, 420503 Russia; fax: (8432) 38-7577; E-mail: yagodina@sci.kcn.ru

²Kazan State University, Kazan, Russia

^* To whom correspondence should be addressed.

Received January 10, 1999; Revision received April 26, 1999

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The optimal conditions for labeling of binase (a bacterial ribonuclease isolated from Bacillus intermedius 7P) with a bromoacetamide spin label have been determined. The label is suitable for probing the dynamics of the protein by electron paramagnetic resonance (EPR). Binase samples specifically labeled at residue His-101 of the active center were prepared by incubation for 48 h at 20°C in potassium phosphate buffer (pH 5.5) containing the bromoacetamide (1:3 protein/label molar ratio). Fluorescence assay showed that the structure of the labeled binase is indistinguishable from that of the native protein.

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KEY WORDS: binase, spin label, EPR, fluorescence assay