13] IMMUNOLOGY OF RIBONUCLEASE 241 



We can also distinguish between preparations of different purity by attach- 

 ing the soluble antigen to erythrocytes by means of tannic acid (Boyden, 

 1951). Antisera to the attached antigen agglutinate such sensitized erythro- 

 cytes. The agglutination can be inhibited by the addition of the soluble 

 antigen to the antiserum (Cinader and Dubert, 1955, 1956; Cinader and 

 Pearce, 1956). The amount of protein required for this inhibition depends 

 on the protein impurities present in a given preparation of antigen, that 

 is on the percentage of total protein effective as inhibitor (Cinader and 

 Pearce, 1956). 



When the antigen is an enzyme, the precipitation of antigen should run 

 parallel with the disappearance of enzyme activity from the supernatants; 

 when antigen can no longer be demonstrated in the supernatant, enzyme 

 activity should also be minimal. A different sequence of events indicates 

 the presence of protein-impurities without enzyme activity (Cohn, 1954). 

 None of the approaches can be safely interpreted, if used alone; each of 

 them should contribute its information to the final correlation of evidence 

 relating to homogeneity. 



The following immunochemical experiments with ribonuclease are con- 

 cerned with the homogeneity of the antigen, but it also forms the continua- 

 tion of studies of antibody as an inhibitor of enzyme (Cinader, 1955, 1957), 



THE INTERACTION OF RIBONUCLEASE 

 WITH ANTIBODY 



Crystallized bovine ribonuclease consists of a major component, the enzyme, 

 and of a minor component which is antigenically distinct from the enzyme. 

 The presence of these two components can be demonstrated by the reaction 

 in agar between antibody to crystallized ribonuclease and chromatographi- 

 cally separated fractions of the crystallized protein (Fig. 1). The arrange- 

 ment in the agar plates of reactants and the zone of precipitation formed 

 between them is shown in Fig. 2. A common zone corresponding to the 

 enzyme runs between all the cups containing antigen and the cups contain- 

 ing the antibody. One chromatographic fraction, that first eluted from the 

 chromatographic column, contains a second antigen which reacts inde- 

 pendently of the zone common to all the fractions and which runs across the 

 main zone formed between ribonuclease A and B and the antiserum. 



Electrophoresis in agar of crystallized ribonuclease followed by diffusion 

 of antibody from lateral channels showed the principal antigen to be in- 

 homogeneous (Cinader, Rondle and Pearce, 1955) and to consist of a fast 

 and a slow moving component; these two components of the enzyme are 

 antigenically indistinguishable since antibody reacts with them in agar with 

 the formation of a continuous zone with two maxima. The minor antigen 

 component present in the fraction first eluted from a chromatographic column 

 migrates with the same velocity as the slower of the two electrophoretic 

 components of the enzyme. 



Qps 



