13] IMMUNOLOGY OF RIBONUCLEASE 243 



With some preparations of crystallized ribonuclease a third electrophoretic 

 component is found to move at a greater velocity than the faster of the two 

 components already mentioned and with one preparation of ribonuclease 

 a fourth component of very low mobility was encountered. These two com- 

 ponents are antigenically identical with the two principal electrophoretic 

 components of the enzyme. 



Ribonuclease A was found to consist of one antigenic species which mig- 

 rated in agar with the same two mobilities as the two components usually 

 observed in the electrophoresis in agar of the crystallized enzyme. The 

 possibility that these two electrophoretic components were artifacts of electro- 

 phoresis could be excluded. Immediately after the electrophoresis was com- 

 pleted, portions of the agar were cut out in positions corresponding to the 

 predetermined areas of highest antigen concentration; these were inserted 

 into fresh agar slides and subjected to electrophoresis. At the end of this 

 second run antiserum was allowed to diffuse through the agar from a median 

 channel ; each component of the enzyme was found to react with the forma- 

 tion of a zone showing one maximum only and hence to react as a single 

 electrophoretic component. 



It is unlikely that a second electrophoretic component was present through 

 contamination from the neighbouring chromatographic component of ribo- 

 nuclease B : we have observed two components not only with a pool of the 

 contents of tubes of effluent corresponding to ribonuclease A, but also with 

 the contents of the tube corresponding to the maximum of the chromato- 

 graphic peak of ribonuclease A (Fig. 1). We cannot exclude the possibility 

 that a second electrophoretic component may have been formed after ribo- 

 nuclease A had been separated chromatographically. 



Supernatants from precipitates formed by varying quantities of ribonu- 

 clease A and constant quantities of antiserum were examined by precipitin 

 test and by diffusion and precipitation in agar. In no case could the co- 

 existence of antigen and antibody be shown by precipitin tests, or the pre- 

 sence of an antigen impurity in the equivalence zone by diffusion in agar. 

 However, the co-existence of enzyme and its antibody could be shown in 

 supernatants from precipitates of ribonuclease A and several antisera, when 

 the supernatants were examined for their power to agglutinate tanned and 

 sensitized erythrocytes and for their enzyme activity. Thus antibodies were 

 encountered which gave rise to soluble complexes of enzyme and antibody 

 in a region which would be identified as the equivalence zone by precipitin 

 tests alone (Cinader and Pearce, 1956). Some antibodies formed in response 

 to repeated intravenous injections with bovine ribonuclease gave rise to 

 nearly 50% of soluble complexes of antigen and antibody. It is clear that 

 true combining ratios of antigen and antibody cannot be directly determined 

 with sera of this nature. We therefore selected for the measurement of com- 

 bining ratios an immune serum which showed an equivalence zone by all the 

 criteria employed. This serum was obtained from a rabbit by immunization 



