Comparison of various immunological methods for distinguishing among mammalian pancreatic ribonucleases of known amino acid sequence

Summary Fourteen mammalian pancreatic ribonucleases of known amino acid sequence were compared by 1 or more of 3 different immunological methods: standard quantitative micro-complement fixation, spot-plate micro-complement fixation, and inhibition of phage inactivation. It was found that, while the results obtained by the 3 techniques were correlated with one another, the standard microcomplement fixation procedure was most versatile, economical of materials, and easiest to execute. The standard MCF technique was more sensitive than the spotplate technique to differences in amino acid sequence. The inhibition of phage inactivation method was more sensitive than the standard method for measuring differences among closely related RNases but proved impractical for amino acid differences over 15%; the MCF method could be extended to at least 30% sequence differences. The standard method, moreover, readily detected the single amino acid difference between dromedary and camel RNases.A linear relationship was found between immunological distance (y) in the MCF test and percent sequence difference (x) which fit the equationy=7x. The strength of the correlation between immunological distance and percent sequence difference is consistent with the proposal that a large fraction of the evolutionary substitutions of amino acids in ribonuclease are immunologically detectable. This could be explained either by a multideterminant hypothesis or by a pauci-determinant hypothesis which says that substitutions occurring outside determinants produce small conformational changes influencing determinant reactivity.This work was supported in part by grants from the National Science Foundation (DEB74-11866A01) and the National Institutes of Health (GM-21509) to A.C.W. and a Fulbright-Hays grant to G.W.W. Part of this work was carried out under the auspices of the Netherlands Foundation for Chemical Research (S.O.N.) and with financial aid from the Netherlands Organisation for the Advancement of Pure Research (Z.W.O.). The followingabbreviations are used in this work: RNase=ribonuclease; MCF=micro-complement fixation.