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A Simple Efficient Procedure for the Preparation of Fragments Corresponding to the Variable-Half and Constant-Half Portions of Bence Jones Protein¹

From the Department of Biochemistry Research, Roswell Park Memorial Institute, ² Buffalo, New York 14203

Abstract

A simple efficient procedure was developed for the preparation of two fragments corresponding closely to the variable-half (V_L) and the constant-half (C_L) portions of Bence Jones protein. The fragments resulted from controlled peptic digestion of a type K Bence Jones protein. The close correspondence of the V_L and C_L fragments to the variable half and the constant half of the Bence Jones protein, respectively, was shown by immunoelectrophoretic analyses and a study of the amino acid compositions of the isolated fragments and indicates that pepsin cleaves Bence Jones protein close to the switch point between the variable half and the constant half. Our results thus support the domain hypothesis. The procedure is based upon our finding that peptic digestion of Bence Jones proteins at 37°C yields only the V_L fragment and some remaining intact Bence Jones protein whereas at 55°C, only the C_L fragment results. Intact type K Bence Jones protein (no prior reduction-alkylation) was digested with pepsin at 37°C for the preparation of the V_L fragment, and at 55°C for the C_L fragment. The fragments were isolated from the respective digests by using in each case a single gel filtration. The yields were markedly high, being 70% for the V_L fragment and more than 60% for the C_L fragment.

Footnotes

¹ This work was supported in part by Grant AI-3962 from the National Institute of Allergy and Infectious Diseases.

² A unit of the New York State Department of Health.