Reaction of Bovine Pancreatic Ribonuclease A with 1,5-Difluoro-2,4-dinitrobenzene

Abstract

Bifunctional reagents have recently been used in several studies (l-4) concerned with the stereochemistry of proteins in solution.The objective was to link proximal pairs of functional groups of a protein molecule by means of bifunctional reagents in order to introduce artificial cross-links stable enough to withstand chemical and enzymatic degradative procedures.The expectation was that only closely spaced groups would be crosslinked, thus affording derivatives in which the location of such bridges could be determined.This would provide information concerning the stereochemistry of a protein molecule in solution.Zahn and Meienhofer (1) have used this approach in studying the stereochemistry of insulin with 1,5-difluoro-2,4-dinitrobenzene.Their results on cross-linking were in agreement with the three-dimensional model of this protein proposed by Linderstrgm-Lang (5).Our work, presented in this paper, deals with the reaction of bovine ribonuclease A with 1,5-difluoro-2,4dinitrobenzene under conditions favoring production of monomeric intramolecularly cross-linked derivatives.It also deals with the purification of these derivatives on Sephadex G-75 and Amberlite IRC-50, and with their characterization in regard to molecular weight, bridge content, and spectral and enzymatic properties. EXPERIMENTAL PROCEDUREMatetials-A commercial preparation of bovine pancreatic RNase, obtained from Mann Research Laboratories (Lot E 1506), was purified by gel filtration on a Sephadex G-75 column in 0.1 N NaHCOa.The purified material consisted of 940/, RNase A as judged by IRC-50 chromatography in 0.18 M phosphate buffer, pH 6.25.It contained 0.11 mole of inorganic phosphate per mole of RNase.1,5-Difluoro-2 ,Cdinitrobenzene (mol wt, 204) was purchased from K and K Laboratories, Inc. To prevent hydrolysis the reagent was stored at 5" in a tightly closed bottle over anhydrous calcium chloride.2,4-Dinitrophenylene-l , 5-RNase