IMMOBILIZED PH GRADIENT FOCUSING OF ALKALINE PROTEINS - ANALYSIS OF THE ISOFORM COMPOSITION OF PURIFIED HUMAN NONSECRETORY RIBONUCLEASES FROM KIDNEY, LIVER AND SPLEEN

Previous studies on the isoform composition of human ribonucleases (RN Aases) have resulted in confusing and inconsistent results, presumably due to methodological problems in electro-focusing of alkaline protei ns. In the present study, immobilized pH gradient (IPG) carrier amphol yte (CA) isoelectric focusing (IEF) and conventional CA-IEF have been evaluated for the analysis of the isoforms of human non-secretory RNAa ses purified from kidney, liver and spleen. CA-IEF proved unsuitable s ince the alkaline RNAase isoforms migrated into the cathode. IPG-CA-IE F, however, resolved the RNAase isoforms and marker proteins in the ba sic region of the gel matrix. The three RNAases had comparable isoform profiles, each with two protein bands with approximate pI values of 1 0.3 and 10.4. Western blotting showed that the two protein bands of ea ch RNAase were immunoreactive (with polyclonal antibodies that recogni ze RNAase), indicating that the protein bands are RNAase isoforms. The present results provide reliable pI data on human RNAase isoforms and suggest that IPG-CA-IEF should be a suitable technique for analysing the isoforms of other alkaline proteins.