Proteolytic Contaminants of Crystalline Ribonuclease

Abstract

Several workers (1-5) have reported that crystalline ribonuclease (6) can hydrolyze proteins, but they have not demonstrated whether this was due to an intrinsic property of the ribonuclease molecule or to the presence of impurities. Schneider (7) showed that the inhibitory effect of ribonuclease on suetime dehydrogenase was apparently due to a contaminant (possibly a proteolyric enzyme) in the crystalline ribonuclease. Kunitz (6) noted the possible presence of a small amount of impurities in the sample of ribonuclease used for solubility tests. Experiments in this laboratory have confirmed the presence of proteolytic activity in every sample of crystalline ribonuclease tested and have shown that all of this activity was due to impurities. EXPERYMF.NTALSamples of crystalline ribonuclease prepared by the method of Kunitz (6) were tested for their ability to digest denatured hemoglobin (8). Several representative assays are given in Table I. Every specimen checked showed some protease activity, and many of the samples also manifested additional protease activity after activation with trypsin, thereby indicating the presence of proteolytic precursors. The total quantity of protease activity and the ratio of protease to nuclease activity, however, varied enormously with the different preparations, showing that at least part of the proteolytic activity was due to impurities. This was confirmed by experiments on the differential destruction of the various activities of the ribonuclease preparations. The effect of pH and of salt concentration on the heat inactivation of ribonuclease and its concurrent protease activity is shown in Tables II and III. The data show that the ratio of nuclease to protease activity was not constant and that under very specific conditions it was possible to destroy all the protease activity while leaving the nuclease activity practically intact. Protease activity is therefore not an inherent property of the ribonuclease molecule.The proteolytic activity of the samples of ribonuclease tested could have been due to contsmination by both chymotrypsin and trypsin, since they were found to clot milk (9) and to hydrolyze benzoyl-l-arginineamidc a (I0). No tests were made for other peptidases. Assuming that one-half of the proteolytic activity was due to trypsin and one-half to chymotrypsin, the median

A Cytochemical Investigation of the Nuclear Changes in the Cyst Cycle of the Ciliate Telotrochidium henneguyi.*

“…The RN-ase solution contained 1 mg of crystalline RN-ase per ml distilled water adjusted to a pH of 6.0. It was heated to 80" in order to inactivate any proteolytic enzymes that could be present ( 11). The DN-ase solution was prepared by adding 1 mg of crystalline DN-ase per ml distilled water adjusted to a pH of 7.0.…”

Nonoxidative Nonproteolytic Enzymes

“…Kleczkowski (119) and McDonald (120) state that the proteolytic activity of crystalline ribonuclease varies enormously in different preparations. A method for the preparation of protease-free ribo nuclease is described (121).…”

Histochemistry of Connective Tissue: I. The Use of Enzymes as Specific Histochemical Reagents

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