The Journal of Biochemistry, Vol. 60, No. 2, 1966 Purification and Properties of RNase T2* By TSUNEKO UCHIDA (From the Department of Biophysics and Biochemistry, Faculty of Science, the University of Tokyo, Bunkyo-ku, Tokyo) (Received for publication, December 17, 1965) In Taka-Diastase, a commercial product of Aspergillus oryzae, have been found two MATERIALS AND METHODS ribonucleases (1), which have been designated Taka-Diastase Powder-Varying samples of Taka- as RNase TI [EC 2.7.7.26, Ribonucleate Diastase powders were kindly donated from Sankyo Co., guaninenucleotido - 2' - transferase (cyclizing), Ltd., Ginza, Tokyo, to which the author expresses Aspergillusoryzae] and RNase T2 [EC 2.7.7. 17, her gratitude. Taka-Diastase A appeared to be more Ribonucleate nucleotido-2'-transferase (cycli suitable for purification of RNase T2 than Taka- zing), Aspergillus oryzae]. Diastase Y (RNase T, rich powder), as described below. The specificity of these two enzymes are DEAE-cellulose was the products of the Brown very characteristic and quite different from Company (20•~l000ƒÊ), the capacity of which was 0.98-0.88 mEq per g. They were more suitable for that of pancreatic RNase [EC 2.7.7.16, batch-wise use and rapid elution in column chromato RNase I-A). RNase T1 specifically hydrolyzes graphy than the products of the Serva Company phosphodiester bonds of guanosine 3'-phos (15-20•~100ƒÊ). phate in RNA via a 2', 3'-cyclic phosphate Sephadex G-75 (40-120ƒÊ, beads type) used for gel intermediate (1, 2). Partially purified RNase filtration was manufactured by Pharmacia Company. T2 was reported to attack preferentially phos Assay for RNase T2-Assay for RNase T2 was phodiester bonds of adenosine 3'-phosphate performed by measuring the absorption at 260 mƒÊ of in RNA and hydrolyze slowly those of other acid-soluble digestion products from the commercial nucleotides (3). RNA according to the assay for RNase T, described On the other hand, previous reports (4) previously (5 ), except that 0.2 M sodium acetate buffer of pH 4.5 was used instead of 0.2 M Tris-HCl buffer suggested the feasibility of analyzing the of pH 7.5. The commercial RNA used for assay was nucleotide sequence in RNA by applying the prepared from Torula yeast and kindly donated by specific cleavages by RNase I-A, RNase T1 Toyo Spinning Company, for which the blank value and RNase T2, provided that RNase T2, was approximately 0.1 at 260mƒÊ. Partially purified when completely purified, would be specific enzyme solution after step 5 was diluted with 0.1% for adenylic acid phosphodiester bonds. gelatin solution to avoid the denaturation of enzyme Therefore, it has long been desirable to due to dilution, when it was used for assay. The elucidate the specificity of RNase T2 using a assay curve of optical density at 260 min was linear sufficiently purified sample of the enzyme. up to increase the value of approximately 0.6. The In the prerent paper the purification protein concentration was determined by measuring the absorption at 280 mƒÊ of the enzyme solution. and properties of RNase T2 will be described The enzyme unit and specific activity were and discussed in comparison with RNase T1. calculated in accordance with the definition used for RNase T1 (5). * Most part of this report was presented at the Assay of RNase T2 for Contaminating Enzyme- annual meeting of the Biochemical Society of Japan, Purified RNase T2 was tested for nonspecific phos November, 1962: J. Japan. Blochem. Soc. (in Japanese), phodiesterase [EC 3.1.4.1] using bis-p-nitrophenyl 34, 407 (1962). phosphate as a substrate (6). A part of tl is work was supported by a grant Paper Electrophoresis-Paper electrophoresis of from the Ministry of Education. RNase T2 was carried out using a Beckmann-Spinco 115 116 T. UCHIDA paper electrophoresis apparatus at 5 mA, 105 volts for alcohol saturated with 0.05N phthalate buffer of 20 hours. Four buffer solutions were used ; veronal pH 5.0. The aqueous phase of hydrolysate was buffer at pH 7.93 , ƒÊ=0.05, phosphate buffer at examined by the paper chromatography in the system pH 5.91 i ƒÊ=0.05, and acetate buffer at pH 4.98 and of tert-amyl alcohol saturated with 0.05N phthalate pH 3.96, ƒÊ=0.05. One gram of bromophenol blue in buffer of pH 6.0, after desalting through a talcum 100 ml. of ethanol containing 10g. of HgCl2 was used column (0.9•~2 em.) and eluting with the mixture of for fixation and staining. N HCl and ethanol (1: 4). Ultracentrifugation-Ultraeentrifugation was per The spots of DNP-amino acids and those for the formed in a Spinco model E apparatus, using a synthetic paper blank were separately eluted from the paper boundary cell and schlieren optics. Sedimentation with 1% NaHCO3 solution at 55-60•Ž for 15 minutes velocity was determined under the same condition* as and subjected to spectrophotometry at 360 mp, assuming that for RNase T1 (7), in order to facilitate the direct a millimolar extinction coefficient of 17.4 for DNP- comparison of both results. Runs were performed at glutamic acid and 17.7 for DNP-ƒÃ-lysine and its relating 59,780 r.p.m. and near 20•Ž. Prior to runs, samples substances. had been concentrated by ethanol precipitation and Passed-through fraction from a talcum column dialyzed against phosphate buffer of pH 6.57 , ƒÊ=0.2. containing free amino acids was dried in vacuo and Sedimentation equilibrium experiment was per submitted to basic amino acid analysis with the use formed as follows**. The cellophane tube containing of a Beckman-Spinco (Model MS) automatic amino purified RNase T2 was buried in Sephadex G-25 to acid analyzer, for estimating the recovery from the concentrate RNase T2 and dialyzed against phosphate amounts of free arginine and making certain of absence buffer of pH6.57, p=0.2. The final concentration of ƒÃ-lysine. of RNase T2 was 9.07 mg. per ml. For these experi Amino Acid Analyses-Salt free preparations were ments, 0.03 ml. of protein solution was used, giving a used for acid hydrolyses, performic acid oxidation and column height of about 0.93 mm. The rotor was run tryptophan determination. The salt free protein at 12,590 r.p.m. and 26.7•Ž for 5 hours after accelera solution in a test tube, of which protein content was tion, in which period equilibrium was certainly determined spectrophotometrically (0.7 to 1.0 mg.), established. was dried in a desiccator over cone. H2SO4 in vacuo Determination of N-Termimal Amino Acid-The N- and the residue was subjected to amino acid analyses terminal amino acid was determined by the DNP- before and after performic acid oxidation. Acid hydro method of Sanger (8). A weighed amount of native lyses of RNase T2 were carried out with 0.5 ml. of glass or performic acid oxidized RNase T2 was dinitropheny distilled 5.7 N HCl at 110•Ž for 32 hours or 72 hours lated in 1.5 ml. of 66% ethanol solution containing in sealed evacuated tubes. Performic acid oxidation 0.5 ml. of 2% DNFB ethanol solution and 25 mg. of of RNase T2 for the determination of half-cystine as NaHCO3, at 37•Ž overnight with shaking in the dark. cysteic acid and methionine as methionine sulfone was To the reaction mixture, which had been acidified performed by the procedure of Hirs (9). The acid with one drop of cone. HCl, was added 2 ml. of hydrolysis of oxidized RNase T2 (2 mg.) was carried ethanol to precipitate the dinitrophenylated protein out for 32 hours. Amino acid analyses of acid and the mixture was centrifuged. The DNP-protein hydrolysates were performed with use of the Beckman- was washed with ethanol (2•~2m1.) and ether and Spinco (model MS) automatic amino acid analyzer*. dried in vacuo. Tryptophan was estimated spectrophotometrically by The DNP-protein was hydrolyzed in a sealed the method of Goodwin and Morton (10). evacuated tube with 1.0 ml. of glass-distilled 5.7 N HCl Carbohydrate Content-The carbohydrate content in at 105•Ž for 16 hours or 5 hours (in the case of the RNase T2 preparation was determined by the DNP-oxidized protein). The ether extract from the phenol-sulfuric acid method of D u b o i s et al. (11). hydrolysate was subjected to two dimensional paper Glucose was used as the standard. Acid hydrolysis chromatography in the system of n-butanol saturated was carried out with N H2SO4 at 100•Ž for 15 hours with 2 N aqueous ammonia and that of tert-amyl in a sealed evacuated tube. Aliquots of hydrolysate were separated in the paper chromatography with a * The author is grateful to Prof. N. Ui and Dr. solvent system of n-butanol : pyridine : water (6: 4 : 3) 0. Tarutani, Dept. of Physical Chemistry, Institute and each spot was detected by aniline hydrogen of Endocrinology, Gunma University, for performing phthalate spray reagent. these experiments. ** The author is grateful to Mrs. M. Kikuchi of * The author is grateful to Mr . S. Horiuchi of this Department, for operation of Spinco model E this Department, for operation of the Beckman-Spinco apparatus. automatic amino acid analyzer. Purification and Properties of RNase T2 117 Gas-liquid partition chromatography was carried out by using a Hitachi-Perkin Elmer Model F-6 Purification Procedurefor RNase T2 assembly fitted with a flame ionization detector*. The purification of RNase T2 was carried Separations were made on the columns of 5 percent Ucon LB 550X on Gaschrom CLH (80-100 mesh), out as follows.