A Method for the Preparation of "protease-free" Crystalline Ribonuclease by Margaret

A method for the preparation of crystalline ribonuclease was described by Kunitz (1) in 1940. Since that time various workers (2-6) have shown that at least some samples of ribonuclease so prepared had, in addition to the ability to degrade ribonucleic acid, the ability to hydrolyze proteins. That the latter was due to contaminants and not to an intrinsic property of the ribonuclease molecule has already been demonstrated (7, 8). The present paper describes a method for the preparation of crystalline ribonuclease free from all measurable traces of proteolytic enzymes. Although the method for the preparation of crystalline ribonuclease described below differs essentially from that of Kunitz in only two major respects, several minor changes have also been made. It therefore seems most useful to describe the complete procedure as it is now used in this laboratory. The first essential change consists of an additional step in the preliminary treatment; namely, boiling the crude ribonuclease preparation in 0.2 saturated ammonium sulfate at pH 3. This destroys all proteolytic and potential proteolytic activity but leaves the ribonuclease molecule practically intact. The second essential change is in the pH of the solution from which the crystals separate. Crystallization is most consistent, and the yields largest, when the pH of this solution is 4.6. The saturated ammonium sulfate is prepared at 20-25°C. (760 gm. of salt per 1000 ml. distilled water). Determinations of pH are made on a test plate by mixing 1 drop of the appropriate 0.01 per cent indicator with 1 drop of the solution to be tested, and comparing the color with that formed by 1 drop of the same indicator with 1 drop of 0.1 M standard buffer of the desired pH. The extent of purification of the ribonuclease in an average preparation is shown in Table I. Twelve such preparations have now been made and assayed. All were tested for their ability to clot milk and to hydrolyze denatured hemoglobin, egg albumin, protamine (salmon), histone (calf thymus), and benzoyl-/arginineamide. In no case was any trace of protease activity detected. Acidified solutions of these preparations containing 0.04 mg. of nitrogen per ml. when left in contact with denatured hemoglobin (8) for 96 hours at 25°C. gave no increase in split products not precipitable with trichloracetic acid. They therefore contained less than 0.002 per cent of proteolytic contaminants (calculated 39