In Taka-Diastase, a Commercial Product of Aspergillus Oryzae, Have Been Found Two MATERIALS and METHODS

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. 2 m., at 191°C and at 165°C. Step 1. CrudeExtract-Five hundred grams of Taka-Diastase powder were well dispersed in 3 liters of distilled water. The suspension EXPERIMENTAL AND RESULTS was centrifuged at 3000 r.p.m. for 10 minutes. Purification of RNase T2 During the centrifugation the powder dis solved almost completely without any special Estimation of RNase T2 Activity in Taka- treatment. If a brown gum-like residue Diastase Powder-The radio of RNases Ti to remained under the black and muddy residue T2 in crude preparation was principally at the bottom of the centrifuge tube, the reflected on the ratio of enzymatic activity whole residue was re-extracted with 2 liters at pH 7.5 to that at pH 4.5. However, the of distilled water. The muddy precipitate was most part of enzymatic activity at pH 4.5 centrifuged off at 3000 r.p.m. for 10 minutes. was responsible for RNase Ti because it The clear, reddish-brown supernatant solution exhibited a considerable activity at pH 4.5 (5 liters) had a conductivity corresponding to {pH 7.5 activity/pH 4.5 activity=5). So, the that of a nearly 0.1 M salt solution. It was amounts of RNase T2 were estimated by adjusted to pH 7.0 with 2 N NaOH. DEAF-cellulose column chromatography of Step 2. Batch-wise Treatment with DEAE- water extract of Taka -Diastase (pH 7.5 cellulose-After about 300 g. of DEAF-cellulose activity/pH 4.5 activity = 2.3, 500 mg./30 ml. were washed with 0.5 N NaOH and then water) according to Taka hashi (5). RNase with water until the pH of the suspension activity at pH 4.5 was separated into three was below 9, they were washed 2 or 3 times

parts : pass-through part, RNase T2 part and to be equilibrated with 0.005 M Na2HPO4. RNase Ti part. RNase T2 part was usually About 2/3 portions of the filter cake composed of two components. The relative (corresponding to 1 /2 weight of the total ratio of activities of the three parts was protein in the crude extract) were added to 10:4:50. However, when the pass-through the crude extract and the suspension was fraction was heated at 80•Ž for 2 minutes at filtered after occasional stirring and standing

pH 1.5, 63 per cent of their activity at pH 4.5 for 20 minutes at room temperature. Pre remained. This heat - stable activity was liminary experiments have shown that the further rechromatographed on DEAE-cellulose adsorption equilibrium between the solution column after desalting by gel filtration with and DEAF-cellulose was accomplished in Sephadex G-25 and found to be completely 10 minutes. The activity remaining in the due to RNase T2. Therefore, the enzymatic filtrate was further adsorbed on DEAE- activity at pH 4.5 derived from RNase T2 in cellulose by repeating this treatment. The Taka-Diastase powder used for this experi passed-through fraction, which was rich in ment could be calculated to be about RNase T2, contained 25-30 per cent of the 16 per cent. The fact that the ratio of total activity at pH 4.5 in crude extract. enzymatic activity at pH 7.5 to that at pH 4.5 When more than 40 percent of the total was fairly variable depending on the lot, was activity at pH 4.5 passed through the column, considered to indicate that the amounts of a considerable part of RNase T1 passed RNase T2 contained in Taka-Diastase were through together with RNase T2, indicating also variable depending on the lot. that the salt concentration in the crude extract was too high to make RNase Ti * The author is grateful to Prof. T. Yamakawa, almost completely adsorb on the column. the Institute for Infectious Diseases, the University of In this case the crude extract had to be Tokyo, for performing these experiments. further diluted with water. Fifty or sixty 118 T. UCHIDA pre cent of the total activity at pH 7.5 was water bath until the temperature of solution nearly recovered from DEAF -cellulose by reached 78•Ž, kept at 78-81•Ž for 2 minutes, elution with 0.35 M NaCl (1.5 liters x 4). and quickly cooled to room temperature in After use, the DEAF-cellulose was regene an ice bath. The fine precipitate in the rated by washing, in turn, with 2 N NaCl, turbid supernatant fluid became larger in size 0.5 N NaOH, and then water. It was quite during the heating. The remaining enzymatic well usable for this batch-wise treatment activity at pH 4.5 was 80 to 95 per cent. This until 5 times regeneration. heat-treated suspension was used in the next

Step 3. Heat Treatment - As shown in procedure. The heat treatment was advan Fig. 1, the RNase T2 activity in crude tageous for inactivating contaminating en preparation rapidly disappeared during the zymes, such as DNase, nucleotidases, and heating at 80•Ž for 2 minutes within the proteases. range of pH 2.5 to pH 6.0, at which range Step 4. Concentration with Ammonium Sulfate contaminating proteins precipitated so much -The protein and enzymatic activity at that RNase T2 could be co-precipitated with pH 4.5 and 7.5 in each supernatant, which them. So the heat-treatment was carried out were equilibrated at pH 6.0 with ammonium as follows. sulfate solutions of varying degree of satura RNase T2 rich fraction was adjusted to tion from 0.1 to 0.9 in final concentration, pH 1.5-1.8 with 2 N HCl. After a small fine were measured. As shown in Fig. 2, RNase precipitate was removed by centrifugation a: T2 activity was precipitable between 0.4 and 3000 r.p.m. for 15 minutes, the turbid super- 0.7 saturation of ammonium sulfate, while natant fluid was poured in a stew pan

(1.5 liters), heated with stirring in a boiling

FIG. 2. Salting-out of RNase T2 with

ammonium sulfate at pH 6.0.

FIG. 1. Effect of pH on the thermostability Each I ml. of RNase T2 preparation after heat

of passed-through fraction from DEAE-cellulose. treatment (1 % soln). was added to 4 ml. of The passed-through fraction in step. 2 was ammonium sulfate solution, in which varying adjusted to each pH and heated to 80•Ž for 2 amounts of solid ammonium sulfate from 0.45 to

minutes in a metal tube for centrifugation. After 0.95 saturation at final were dissolved with 0.1 M

cooling and removal of precipitate by centrifuga citrate buffer at pH 6.0. After standing overnight tion, the protein (-) and RNase activity at at room temperature, the protein (-) and RNase activity at pH 4.5 (-•ü-) and pH 7.5 pH 4.5 (-•ü-) and pH 7.5 (----) in each supernatant were measured. (---- ) in supernatant were measured. Purification and Properties of RNase T2 119

RNase T1 activity, above 0.7 saturation and 20 ml. head of 0.005 M Na2HPO4 solution was most part of inactive protein, below 0.4 applied and a nonlinear elution gradient was saturation. Although RNase T2 was precipi started. The mixer flask contained l liter of table within the range of saturation of 0.005 M Na2HPO4 and the reservoir contained ammonium sulfate solution from 0.4 to 0.7, 2 liters of 0.25 M NaCl containing 0.25 M the amount was so small and RNase T2 in NaH2PO4. Fractions of approximately 10 ml. the heat-treated suspension was so dilute that were collected, and the flow rate was appro RNase T2 activity was difficult to be com ximately 3.5 ml. per minute. As shown in Fig. 3, the enzymatic activity was separated pletely precipitated, especially in a large scale into three fractions ; the first two peaks show preparation. Therefore, this procedure was ed both RNase T2 activity and were designated used merely to concentrate all enzymatic activity in heat-treated suspensions and an as RNase T2-B and RNase T2-A in order of elution, whereas the last peak was RNase Tt attempt to separate RNase T2 and RNase Tt obtained as a by-product. Usually the by the process was given up. In further amount of RNase T2-A exceeded that of experiments the concentration of RNase T2 RNase T2-B. The total activity at p144.5 of with ammonium sulfate was carried out as RNase T1 and that of RNase T2 obtained follows. here were almost equal. The heat-treated suspension was adjusted Step 6. Alcohol Fractionation-Figs. 4a and to pH 6.0 with 2 N NaOH, and brought to 4b show the results of alcohol fractionation of 0.4 saturation with ammonium sulfate by the RNase T2 activity in crude extract and in addition of 300 g. of the solid salt per I liter. RNase T2-A fraction at step 5, respectively. After 30 minutes at room temperature, the flocculent precipitate was separated by centrifugation at 3000 r.p.m. for 20 minutes. The supernatant was adjusted to pH 4.0 with 2 N HCl to achieve complete precipitation of RNase Tl activity, and brought to 1.0 saturation by the addition of 390 g. of solid

ammonium sulfate per 1 liter. The turbid suspension was placed in a cold room overnight. The brown precipitate (containing RNase T2 and a part of RNase TI) was collected by centrifugation at 8000 r.p.m. for 20 minutes, dissolved into a small amount of 0.1 M citrate buffer at pH 6.0 and then dialyzed against tap water overnight. Step 5. DEAE-cellulose Column Chromato

graphy I-The dialyzed RNase T2 rich solution FIG. 3. Chromatogram of RNase T2 prepara was adjusted to pH 7.0-7.5 by addition of tion after concentration with ammonium sulfate

0.5 M Na2HPO4 to a final concentration of on a column of DEAE-cellulose. 0.005 M. Column ; 2.8 X 50 cm. Load ; about 8-10g. of RNase T2 preparation after concentration by This enzyme solution (about 750 ml., addition of ammonium sulfate and then dialyzation. containing 8-10 g. of protein) was loaded on Elution ; gradient from 0.005M Na2HPO, to a column of DEAE-cellulose (2.8•~50 cm) that 0.25M NaH2PO4 (containing 0.25M NaCl) with had been previously washed with 0.5 N NaOH all liter mixing chamber, at flow rate of approxi and water and equilibrated with 0.005 M mately 3.5 ml. per minute. Fraction ; 10 ml.

Na2HPO4. Then two 20 ml. portions of per fraction. Protein concentration (---) and 0.005 M Na2HPO4 were used to wash the last enzymatic activity at pH 4.5 (-•ü-) and pH 7.5 bit of enzyme solution onto the column. A (---- ) were measured. 120 T. UCHIDA

RNase T2 activity was completely precipitated RNase T2-A fractions were combined and between 35 per cent and 52 per cent ethanol brought to 35 per cent ethanol solution (v/v) within the range of protein concentration of with 99.5 percent ethanol, the temperature 300 ƒÊg. to 10 mg./ml. solution at -2-+5•Ž, being maintained below 5•Ž in an ice bath. and any protein other than RNase T2 was After standing for 20 minutes at 0--2•Ž, the hardly precipitated in this range of ethanol solution was centrifuged at 12,000 r.p.m. for concentration. No loss of RNase T2 activity 20 minutes at -2•Ž. The supernatant fluid was observed below 5•Ž in this procedure. was brought to 52 per cent ethanol solution

RNase T1 and the most part of colored (v/v) with 99.5 per cent ethanol at temperature impurities were precipitated by ethanol of below 5•Ž as above. A large amount of above 52 per cent. Therefore, this procedure crystals of NaH2PO4 precipitated out together was advantageous in elevating the specific with the precipitate of RNase T2. They were activity of RNase T2, and in separating collected by centrifugation at 12,000 r.p.m. for RNase T1 from RNase T2, even when RNase 20 minutes, dissolved in a small amount of T1 had been contained in the RNase T2 0.1 M citrate buffer at pH 6.3 and dialyzed fraction. So the alcohol fractionation was against cold distilled water in a cold room carried out as follows. overnight. The recovery of enzymatic activity was about 80 percent. About 10 percent of enzymatic activity was additionally recovered from 52-60 percent ethanol fraction with colored impurities, especially in a large scale

preparation. The specific activity was raised to 30. This value was variable within the. range of 15-30 depending on the lot of Taka- Diastase powder. If the specific activity was below 25, refractionation with ethanol had to be carried out as described above, so that the activity of RNase T2 after the next

procedure may be sufficiently high. RNase T2-B fractions were fractionated

quite similarly to RNase T2-A fractions. Step 7. DEAE-cellulose Column Chromato

graphy II-The faint yellow dialyzed solution of RNase T2-A was adjusted to pH 7.0-7.5 by addition of 0.5 M Na2HPO4 to a final concentration of 0.005 M. This enzyme solution (about 15 ml., con taining 50-100 mg. of protein) was applied on a column of DEAE-cellulose (1.2 x 18 cm.)

previously equilibrated with 0.005M Na2HPO4 . FIG. 4. Ethanol fractionation of crude extract Then two 5 ml. portions of 0.005 M Na2HPO4 (a) and RNase T2 fraction obtained on a were used to wash the last bit of enzyme DEAE-cellulose column chromatography (b). solution onto the column. A 5 ml. head of Each I ml. of RNase T2 preparation was 0.005M Na2HPO4 solution was applied and added to 4m1. of cold aqueous ethanol at 0-5°C, a nonlinear elution gradient was started. in which the varying amounts of ethanol from The mixer flask contained 280 ml. of 0.005M 10% to 80% at final concentration were included. Na2HPO4 and the reservoir contained 600 ml. After standing for an hour at -2•Ž, the protein of 0.15 M NaCl containing 0.15 M NaH2PO4 . concentration (-) and enzymatic activity at Fractions of approximately 5 ml. were col pH 4.5 (-0-) and pH 7.5 (----) in supernatant were measured. lected, and the flow rate was approximately Purification and Properties of RNase T2 121

1 ml. per minute. RNase T2-A was eluted as A procedure similar to the above was a single peak with respect to the enzymatic also used for the purification of RNase T2-B. activity as well as to the protein concentration The results of a typical preparation are (Fig. 5a). However in some cases, a small summarized in Table 1. By this procedure peak of RNase T2-B was eluted ahead of 5-10 mg. of the colorless and pure RNase RNase T2-A peak, as shown in Fig. 5b. The T2-A were prepared from 500g. of Taka- RNase T2-A fractions of specific activity above Diastase powder and the total purification 50 were collected with the recovery of 50- was about 900-1100-fold. This preparation 60 per cent, dialyzed exhaustively against contained no other enzymatic activity, such several changes of cold distilled water and as phosphodiesterase. RNase T2-B was also stored in a deep-freezer. obtained in the same purity and the tame

yield as RNase T2-A by this procedure. However, the lots of Taka-Diastase had to be selected for success in purification of RNase T2. The lots which showed a large ratio near 5 of enzymatic activity at pH 7.5 to that at pH 4.5, such as Taka-Diastase Y, or which had only a small activity at pH 4.5

(below 600 x l03 units/500 g. Taka-Diastase), were unsuitable as starting materials, for they showed not only a poor yield of RNase T2 but also no raise of the specific activity above 40-50, even though the purification

procedures of steps 6 and 7 were repeated several times. In such cases RNase T2 was successfully

purified on gel filtration by Sephadex G-75. Lyophilized RNase T2 preparation, which was found to show a single peak on DEAE- cellulose column with the specific activity of 50, was dissolved in 2 ml. of 0.02 M NaCl, applied on a column of Sephadex G-75

(1.5 •~ 70 cm.), and eluted with 0.03 M citrate buffer pH 6.3. Fractions of approximately 2 ml. were collected, and the flow rate was approximately 4 ml. per 5 minutes. As shown in Fig. 6, about 10 per cent of the high FIG. 5. Chromatograms of RNase T2 pre molecular contaminants was observed in parations after ethanol fractionation on a column addition to the main peak with the specific of DEAE-cellulose. activity of above 60. When RNase T2 with a) and b) show that the separation of RNase the specific activity of 30 was applied on a T2-A and T2-B in step 5 (Fig. 3) was successful column of Sephadex G-75, these high-molec in a), but not in b). Column : 1.2 X 18 cm. Load : ular contaminants increased to a half of the about 15m1. (containing 50-100mg. protein) of main peak, as measured by the absorbancy RNase T2 preparation after ethanol fractionation. at 280 mu, and accompanied with about 1/4 Elution : gradient from 0.005 M Na2HPO4 to 0.15 M of the total activity. The nature of these NaH2PO4 (containing 0.15M NaCl) with a 280 ml. mixing chamber, at flow rate of approximately contaminants has not yet been studied. 1 ml. per minute. Fraction : 5 ml. per fraction. Properties of RNase T2 The protein concentration (-) and RNase activity at pH 4.5 (-0-) were measured. The purified RNase T2 was shown to be 122 T. UCHIDA

TABLE I Purification of RNase T, from 500 e. of Taka-Diastase

1) After step V, only the result for RNase T2-A is shown. 2) Figures in parentheses indicate activities derived from RNase T2 (assuming that its content is 16% of total activity at pH 4.5) and the specific activities calculated from it, respectively.

homogeneous in chromatography on DEAE- cellulose, gel filtration on Sephadex G-75, paper electrophoresis, sedimentation and N-terminal amino acid analysis. Paper Eletrophoresis-RNase T2-A moved to the anode much slower than RNase Tl above pH 4.98 , and contrary to RNase T, to the cathode at pH 3.96. RNase T2-B travelled nearly as much as RNase T2-A at all pH's examined. RNase T2-A was regarded as homogeneous at all four pH's tested, that is, pH 7.9s, pH 5.9, , pH 4.98 and pH 3.96. The

TABLE II The M.ou'ng Distan:e of RNase T2 in Paperelectrophoresis

FIG. 6. Gel filtration by Sephadex G-75 of RNase T2 preparations with specific activity of 50 (a) and 30 (b). Column ; 1.5 X 70 cm. Load ; 5.1 mg. and 6.7 mg. of RNase T2-A in 2 ml. of 0.02M NaCl in (a) and (b) respectively. Elution ; 0.03M citrate buffer pH 6.3, at flow rate of approximately homogeneous in chromatography on DEAE-cellulose, gel filtration on Sephadex G-75, paper electrophoresis, sedimentation and N-terminal amino acid analysis.Paper Eletrophoresis-RNase T2-A moved to the anode much slower than RNase T1 above pH 4.98 , and contrary to RNase T1 to the cathode at pH 3.96. RNase T2-B travelled nearly as much as RNase T2-A at all pH's examined.RNase T2-A was regarded as homogeneous4 at ml. all fourper pH's tested,5 minutes. that is, pH 7.93, FractionpH 5.91 , pH 4.98; and2 ml. pH 3.96.per TheTABLE fraction. IIThe A ov'ng Distan:e of RNase1) T2inacetate Paperelectrophoresis buffer, to=0.05 The protein concentration (-), RNase activity . 2) phosphate buffer,ƒÊs=0.05 at pH 4.5 (-•ü-) and NaCI concentration (---) 3) veronal buffer, ,u=0.05 were measured. Purification and Properties of RNase T2 123

moving distance of RNase T2-A at each pH under this experimental condition is shown in Table II as compared with that of RNase TI, from which value the isoelectric point of RNase T2 was estimated graphically to lie around pH 4.9. Ultracentrifugation-Both RNase T2-A and RNase T2-B showed a single symmetrical boundary on the ultracentrifugation analyses, as shown in Fig. 7. One run on RNase T2-A was made at the concentration of 3.94mg. per ml. and two runs on RNase T2-B were made at the concentrations of 3.03mg. per ml. and 6.25mg. per ml. If a partial specific volume (v) of 0.73 was assumed, the s20,w values were calculated Fin. 7. Sedimentation pattern of RNase

T2-A. to be 3.44S for RNase T2-A, 3.53S and 3.2sS The concentration of RNase T2-A was 0.394%. for RNase T2-B respectively. The s20,w

Runs were performed at 59,780 r.p.m. and 21•Ž. values for RNase T2-A was calculated to be This picture was taken at 49 minutes after reaching 3.05S, assuming a partial specific volume of full speed. 0.695 (that of RNase I-A) according to

TABLE III Determination of N-terminal Amino Acid of RNase T2

1) These values were calculated on the basis of the assumption that the total amount of ƒÃ-DNP-Lys

(ƒÃ-DNP-Lys+spot 2) was 23 moles per mole enzyme.

2) Estimated from the ratio of the total amounts of ƒÃ-DNP-Lys to the weight of protein weighed at the start of the sequential reaction processes (see below).

3) Estimated on the basis of the amounts of free arginine obtained by amino acid analysis of the passed-through fraction from a talcum column.

4) These values show the total recovery throughout the sequential procedures, namely, oxidation with performic acid followed by lyophilization, dinitrophenylation, acid hydrolysis, extraction with ether and chromatography.

5) This value shows the total recovery resulted from the sequential procedures, without the procedure of performate oxidation. 124 T. UCHIDA

Rushizky and Sober in good agreement that the total amounts of ƒÃ-DNP-lysine

with their results (12). RNase T2-A and RNase (ƒÃ-DNP-lysine +spot 2) was 23 moles per mole T2-B were both homogeneous and had no enzyme because of the absence of free difference from each other in the molecular ƒÃ-lysine and the recovery of N-terminus in size. From the above three observed values, this procedure was just the same as that of s0 20 , w of RNase T2 was estimated graphically ƒÃ-lysine. The recovery estimated for ƒÃ-lysine to be 3.61 S. demonstrates good agreement with the The purified RNase T2 was prepared in recovery estimated on the basis of the too small a quantity to measure the diffusion amounts of free arginine found in aqueous

coefficient or viscosity. So, the molecular phase of the acid hydrolysate of DNP- weight was determined by sedimentation oxidized RNase T2. equilibrium. The plot of In C versus N2 Enzymatic Activiy-One mg. of the most

was linear, indicating homogeneity with purified enzyme possessed about 14 x 102 units respect to molecular weight. If a partial of activity and the specific activity of the specific volume (v) of 0.73 was assumed, the best preparation was 70, with was about molecular weight of RNase T2 would be one tenths of that of RNasc T1 (13). 36,200. Stability-As shown in Table ‡W, RNase N-Terminal Amino Acid-In the ether T2 is somewhat less stable than RNase T1,

extract of acid hydrolysate of DNP-RNase but is fairly stable as compared with most T2 or oxidized RNase T., only DNP-glutamic of other enzymes. It was most stable at acid or DNP-glutamine was found, but in around neutral pH when kept at room the aqueous phase two unknown spots, spots temperature and also when heated. It was I and 2, were detected besides DNP-ƒÃ-lysine. heated at 90•Ž for 5 minutes at pH 6.0 without The amounts of DNP-derivative obtained loss of activity, but it lost the activity fairly from each spot are shown in Table ‡V. rapidly above 90•Ž. At weak alkaline pH it Spot 1 showed the same Rf values as suffered little inactivation at room tempera that of DNP-cysteic acid on paper chromato ture, in contrast to the instability of RNase

graphy, but migrated with DNP-derivatives T1 at alkaline pH (14). However, at acidic of neutral amino acids on paper electro pH region RNase T2 was rather less stable

phoresis in the system of pyridineacetate than RNase T1. buffer of pH 3.5 at 12.5 volts/cm. for 2 hours. About 80% of RNase T2 activity was Spot 2 was detected around DNP-arginine found to be stored frozen for several months on paper chromatography, and migrated at neutrality unless freezing and thawing of slightly faster than DNP-lysine on paper the enzyme solution was repeated. On electrophoresis in the system of N ammonia lyophilization only about 15% inactivation at 19 volts/cm. for 2 hours. Furthermore at pH 6.0 was observed.

spots I and 2 were both ninhydrin positive, pH Optimum-The pH-activity curves for indicating that these derivatives possessed RNase T2 with high-molecular-weight yeast free NH2-groups and did not arise from the RNA and commmercial RNA as substrates N-terminal amino acid. are shown in Fig. 8. RNase T2-A and In the case of prolonged hydrolysis of RNase T2-B are not distinguishable in this DNP-protein, the amount of spot 1 increased respect either. These enzymes were most and that of spot 2 decreased. It is con active in 0.05 M citrate-phosphate buffer at cluded from the above results that spot 1 is pH 4.5, with 50% of maximal activity an artifact resulted from hydrolysis and remaining at pH 3.6 and 5.4 for the com spot 2 is a peptide containing ƒÃ-DNP-lysine. mercial RNA, and at 3.7 and 5.05 for hig Therefore glutamic acid or glutamine molecular-weight yeast RNA, which was was found as the only N-terminal amino prepared from pressed cakes of bakers' yeast acid of RNase T2 and amounted to about by treatment with detergent (15) followed 0.8 moles per mole of the enzyme, assuming by the phenol treatment. However RNase Purification and Properties of RNase T2 125

TABLE IV Stability of RNase T2

To 0.4 ml of each buffer solution was added 0.1 ml of RNase T2-A (0.3 mg./ml.). After standing for arbitrary time at room temperture, or heating to 80•Ž for 5 minutes in a sealed tube and cooling in an ice bath, each solution was diluted to 25-times with 0.05 M citrate-phosphate buffer of pH 4.5 containing

0.05% gelatin and the enzymatic activity was determined immediately. The enzymatic activity of the same RNase T2-A solution, measured as soon as it was diluted to 125-times with 0.05 M citrate-phosphate buffer of pH 4.5, was set to 100%. The following buffer solutions were used; 1) 0.1 M HCl, 2) 0.05 M citrate-phosphate buffer, 3) 0.05 M Na2CO3-NaOH buffer, 4) 0.01 M HCl, and 5) 0.05 M Tris-HC1 buffer.

T2 exerted abont 1.75 times as high activity at pH 4.5 on the commercial RNA as that on the high-molecular-weight yeast RNA. The difference between the enzymatic activ ity upon the commercial RNA and that upon the high-molcular-weight yeast RNA was most pronounced at around pH 6.0. The activity at pH 7.5 was about 7.5% of the maximal activity. The kinds of buffer, citrate-phosphate or acetate, did not effect the enzymatic activity. Activators and Inhibitors-As shown in Table V, the enzymatic activity of RNase T2 was inhibited strongly by Cu++ and slight ly by Mg++ and Ca++, in good agreement with the previous results for partially purified RNase T2 of Naoi-Tada, Sato-Asano and Egami (3). However, in contrast to their results, RNase T2 was inhibited also by FIG. 8. pH dependence of the RNA digesting Zn++ as well as RNase TI. This discrepancy activity of RNase T2-A. can be explained by the fact that Zn++ at Citrate-phosphate buffers of 0.05M were used 10-3M could inhibit RNase T2 only when it in all region of pH tested. Commercial RNA (--•œ--) and the high-molecular-weight yeast RNA was added to the enzyme before addition of substrate, as shown in Fig.9. Furthermore (-•ü-) were used as substrates. The maximum activity for each substrate was taken set as 100%. most purified RNase T2 was not so clearly 126 T. UCHIDA

TABLE V Activators and Inhibitors

Activity was measured at pH 4.5 with various inhibitors and activators in stead of EDTA, according to assay for RNase T2, but without EDTA. Addition of RNA, that is, the start of reaction, was per formed after preincubating enzyme and reagent for 20 minutes. 1) Determined by Takahashi (14).

activated by EDTA as the partially purified analyses were performed ; 32 hours' and 72 enzyme and by histidine as RNase T1. hours' hydrolysates of native RNase T2, and RNase T2 was more easily inhibited by salt 32 hours' hydrolysate of performic acid- concentrations above 0.1 M than RNase oxidized RNase T2, for making certain of T1 did. the existence of methionine residue. In the

Ultraviolet Absorption Spectrum-The UV chart of chromatogram of 32 hours' hy absorption spectrum of RNase T2-A in water drolysate with use of an amino acid auto is shown in Fig. 10, which has a maximum matic analyzer, this enzyme was shown to absorption at 281 mƒÊ, a minimum one at contain all of the common amino acids and 252 mp and two shoulders around 278 mp no unknown substance, such as particular and 290 mƒÊ. The latter shoulder appeared amino acid and amino-sugar. The recoveries to show the high content of tryptophan in in dry weight calculated with the assump RNase T2. The enzyme solution of one mg. tion of O.D.°80!,~=1.95 were 89.5%in 32 hours'

per ml. showed the optical density of 1.99 hydrolysate and 87.0%in 72 hours' hydrolysate, at 281 mg. respectively. The recovery of amino acids

The absorption spctrum of RNase T2-B did not increase with increasing time of acid was identical with that of RNase T2-A. hydrolysis and non-degraded oligo-peptide Amino Acid Composition-Three amino acid was not found in 32 hydrolysate. Therefore, Purification and Properties of RNase T2 127

Table VI. The molar ratio of each amino acid in 32-hour and 72-hour hydrolysates was calculated taking the quantity of arginine residue as 1. The values for labile amino acids, such as serine, threonine and tyrosine, were corrected for destruction by linear extrapolation to zero time, and those for saline, leucine, isoleucine and proline, were corrected for incomplete liberation from the 72-hour values only. The results for cystine and methionine were given as averages of the molar ratio calculated as Arg=1 and that calculated as Phe=2.55 from the hy drolysate of performic acid oxidized RNase T2. Thus methionine was found to be an amino acid with the least amount in the enzyme and the molar ratio of amino acids FIG. 9. Inhibition of RNase T2 with various were recalculated. as methionine residue concentrations of Zn++. Reaction mixture con equal to 1. tained 0.4 units of RNase T2-B, various amounts of Zn++ from 10-1M to 10-5M at final, and 3 mg. Ammonia was found to increase with of commercial RNA in 1 ml. of 0.05 M citrate- increasing time of hdrolysis and the value phosphate buffer. In one case, RNA was added was corrected by extrapolating to zero time. to enzyme solution with Zn++ at the same time The difference between the corrected value (--•--) and in the other, after addition of Zn++ and 32-hour value corresponded to the and standing for 30 minutes (-0-). destructed amounts of labile amino acids. But the result for amide content obtained with the acid hydrolysate by extrapolation was considered to be a reference value and the final analysis of amide content remains to be performed by other method. As to tyrosine and tryptophan, spectro photometrical determination was carried out in parallel with chromatographical analysis. The ratio of tyrosine to tryptophan was determined to be 2.01. The tyrosine content was determined simultaneously to be 14., moles per mole of the enzyme, for which the molecular weight cf 36,200 was assumed from the result of the sedimentation equi librium. This result was in good agreement FIG. 10. Absorption spectrum of RNase T2-A. with the tyrcsine content obtained with the 370 ƒÊg. enzyme in ml. of water with 10 mm. light acid hydrolysates of the native protein. path. From the integral numbers of the amino acid residues in the last column of Table the poor recoveries of amino acids appeared VI, the molecular weight for RNase T2 was not to be due to the incompleteness of acid calculated to be from 36,229 (containing no hydrolysis and it was suggested that RNase amide) to 36,167 (containing 63 moles of T2 contained some substances in addition to amide per mole of the enzyme), which was amino acids. in good agreement with the molecular The analytical results are summarized in weight obtained by sedimentation equi 128 T. UCHIDA

TABLE VI Amino A-id Composition of RNase 7;

1) The amounts of cysteic acid and methionine sulfone were expressed as mean values between the molar ratio calculated as Arg= 1.0 and another ratio as Phe=2.55. 2) The results for the labile amino acids (Ser, Thr) were corrected by extrapolating to time 0. 3) The value of 72 hours hydrolysate only. 4) Extrapolated to time 0. 5) Determined spectrophotometrically.

librium method. From this analytical result were somewhat variable in each prepartion. of amino acid composition, the partial The carbohydrate components were found specific volume of RNase T2 was calculated to be mainly hexoses in paper chromato to be 0.717. graphy of acid hydrolysate of RNase T2-A Carbohydrate Content-As it was suggested and amino sugar was not detected by amino from the poor recoveries on the amino acid acid analysis in the hydrolysates of both analyses that RNase T2 contained some RNase T2-A and T2-B. Furthermore, it was substances in addition to amino acids, the observed by gas-liquid partition chromato carbohydrate content was determined. RNase graphy that RNase T2-A contained mannose, T2-A was found to contain from 12 to 15% glucose and trace amounts of galactose and of sugar in glucose equivalent. The values xylose, while RNase T2-B contained mannose Purification and Properties of RNase T2 129

TABLE

‡Z Physical and Chemical Properties of RNase T2

DISCUSSION By the procedure described above, the most highly purified RNase T2 can be pre pared, though the yield is apparently not so good as described in the procedure by Rushizky and Sober (12). Since RNase T2 is easily inactivated by dilution of the protein solution as mentioned above, it is required for preparing in a good yield that RNase T2 is kept in as high con centrations as possible throughout the process of purification. Attempts to prepare RNase T2 from "RNase T2 fraction" obtained on the way of RNase T1 purification from more FIG. 11. Carbohydrate contents of various diluted Taka-Diastase solution (eluted be RNase T2-A preparations. tween 0.02 M and 0.1 M NaCl concentration from DEAE-cellulose in batch-wise treatment) and galactose as main components and a (16), ended in a failure because the enzyme small amount of glucose and a trace of solution was highly diluted and no effective xylose. method to concentrate it was devised. Also, The relation between the carbohydrate in the procedure described above, a large content and the specific activity of RNase loss of enzyme in a step of concentration by T2-A preparation was followed in the process addition of ammonium sulfate was observed. of purification. As shown in Fig. 11, the In order to carry out more effective purifica carbohydrate content came near to a definite tion, it is desirable to improve this step by value at the specific activity of about 70. looking for the more efficiently concentrating Physical and chemical properties of reagent than ammonium sulfate or specific RNase T2 are summarized in Table ‡Z, as adsorbents for RNase T2, such as acid clay compared with those of RNase T1 (13). used in RNase T1 purification (16). Cation 130 T. UCHIDA .

exchangers, such as IRC 50 or CM-cellulose spectrum also indicates that of a typical were examined for use in batch-wise treat. protein. However, the presence of carbo ment of RNase T2, in which most of RNase hydrate components above 10% was con T1 passed through the exchanger and only firmed in pursuit of the origin of poor RNase T2 activity was retained on the recoveries in the amino acid analyses. The exchanger. Such attempts, however, were possibility that these carbohydrate com unsuccessful owing to the weak affinity of ponents are contaminants of RNase T2, may RNase T2 towards the exchangers. be excluded in view of the result that the In DEAE-cellulose column chromato carbohydrate content of RNase T2 prepara graphy with the suitable gradient system, tions converged to a definite value as the RNase T2 was always separated into two purification proceeds. Though the carbo fractions, that is, RNase T2-A and RNase hydrate composition of RNase T2-A is T2-B, regardless of applying directly a water somewhat different from that of RNase T2-B, extract from Taka-Diastase or RNase T2 the specific activities of both fractions are solution after heat-treatment in acidic pH. quite the same, indicating that these carbo Therefore, a double peak of RNase T2 is not hydrate components are not essential at least considered as an artifact resulted from acid for . the enzymatic activity. Taka-amylase treatment as previously suggested by Rushi [EC 3.2.1. l prepared from the same source, zky and Sober (12 ). No difference can be Taka-Diastase (18), and RNase B from observed between RNase T2-A and T2-B con bovine pancreas, eluted ahead of RNase A cerning the enzymatic properties, such as on column chromatography (19), are cited specific activity, pH optimum, and effects of as other enzyme known as a glycoprotein. activators and inhibitors. RNase T2-A is In both cases, the oligosaccharides were also indistinguishable from RNase T2-B in reported to be attached at aspartic acid or the properties as a protein, such as molecular asparagine residue by a single covalent bond* weight, UV absorption spectrum, N-terminal (19 ), and not to affect the enzymatic activity. amino acid, and amino acid composition. It remains to be elucidated whether RNase Therefore the supposition by Rushizky T2 is also identified as a glycoprotein linked and Sober that the double peak obtained by a covalent bond between protein and from the DEAE-cellulose chromatography of carbohydrate moieties, or the carbohydrate a water extract may be due to the complex components are only wrapped up in a folding formation of RNase T2 with RNase T1 (12), structure of protein. cannot be recognized. It appears that one Our RNase T2 preparations are com and the same enzyme protein exists in some parable to the partially purified enzyme as different situation, rather than the presence previously reported (3), with respect to the of two different enzyme proteins. In fact, pH optimum, the behavior toward various the difference between the two fractions was inhibitors and activators, fractionation with observed in their carbohydrate components. ammonium sulfate, chromatography on Several lines of evidence drawn from the DEAE-cellulose, and mobility on paper- or above experiments, demonstrate that the zone-electrophoresis. Furthermore our pre most purified RNase T2-A and T2-B are parations are more similar to those obtained quite homogeneous enzyme and never a by Rushizky and Sober (12) in detail, mixture of several enzymes, although the that is, in respect to the slight activation by specificity is very wide unlike RNase Ti, EDTA, the high ratio of enzymatic activity at as will be reported in the succeeding paper pH 4.5 and 7.5, and the sedimentation co (17). The molecular weight of RNase T2 efficient. However the ratio of the absorption estimated from sedimentation equilibrium shows a good agreement with the minimum * Anai , M., Ikenaka, T., and Matsushima, Y., molecular weight calculated from the amino presented at the annual meeting of the Chemical acid composition. The UV absorption Society of Japan, April, 1965. Purification and Properties of RNase T2 131

tion at 280 mp and 260 my in the ultraviolet in RNase I-A, while methionine residue is absorption curve supports that our prepara present in the latter and absent in the former. tions are the most purified RNase T2. 3) It was analyzed to possess 11 residues of

The specific activity of the pure RNase half cystine, which value is relatively low for T2 was observed to be 14 x 102 units/mg. the large molecular weight. However the enzyme, using the commercial RNA (mean presence of free SH-groups has not been nucleotide units=25) as a substrate. However confirmed. 4) The basic amino acids form this value is variable depending on the nature about 10 percent of the total amino acid of RNA used for assay. In fact, the RNase residues. Especially RNase T2 is like RNase T2 activity measured by using the high- I-A rather than RNase T, with respect to a molecular-weight yeast RNA, dropped to higher content of lysine, corresponding to about 4/7 of the above-mentioned value. two thirds of total basic amino acids. 5) It Therefore, the low-molecular compound, of is similar to RNase Ti in being more abundant which molecular structure is well known, in glycine residues than in alanine residues. 6) It is relatively abundant in proline residues. such as nucleotide cyclic phosphate, should be used as a substrate for the exact deter Consequently, amino acid composition of RNase T2 is quite different from that of mination of RNase T2 activity. Recently the RNase T1. assay for RNase T2 measured by using the Preliminary experiments* showed that uridine 2',3'-cyclic phosphate as a substrate the activity of RNase T2 appeared in the has been established (20). By this assay, culture medium after the cell growth of the specific activity of RNase T2 was deter Aspergillus oryzae reached the stationary phase, mined to be 4.2•~102 standard units/mg. whereas RNase Ti activity was secreted out enzyme (21). One unit as defined in this of the cell in the logarithmic phase of the paper is found to correspond to about 0.30 standard units (21). growth. Therefore RNase T2 is regarded as an endocellular enzyme and RNase T1 as an As compared with RNase Ti and RNase endocellular enzyme and RNase T1 as an I-A, RNase T2 is characterized by a larger exocellular enzyme. It is not considered that molecular weight (approximately three times) a fragment of RNase T2 is released out of the and the presence of some neutral sugars in cell as RNase T1, because, as mentioned the molecule. The isoelectric point of RNase above, the amino acid content of both enzymes T2 was estimated to lie around pH 5. is quite different, and moreover RNase T2 Therefore RNase T2 is not a so acidic protein gave no precipitate with anti-RNase Ti serum as RNase Ti. RNase T2 possesses 1 mole of and exerted no inhibition on the RNase Ti- glutamic acid or glutamine residue at the antibody precipitin reaction* (14). N-terminus, unlike RNase T, (alanine) (22). So it seems highly probable that RNase T2 SUMMARY is also composed of a single polypeptide chain 1. A method for the purification of RNase as well as RNase Ti and RNase I-A, in spite T2 [EC 2.7.7. 17] is described, which consists of its, larger molecular weight. In respect to of water extraction, batch-wise treatment with the amino acid composition, RNase T2 is DEAE-cellulose, heat treatment, concentration characterized as follows; 1) It contains six with ammonium sulfate, DEAE-cellulose histidine residues per molecule, i, e., twice as column chromatography, ethanol fractionation many as it is the case in RNase Ti (13). and DEAE-cellulose column chromatography This fact is noteworthy, for some of the with a milder elution gradient system. histidine residues might be involved in the 2. By this procedure, RNase T2 was catalytic function of ribonucleases. 2) It purified 900-1100-fold with a yield of about possesses both tryptophan (7 residues) and 10% (15-20 mg. with combined RNase T2-A methionine (one residue). Tryptophan residue * Uchida is present in RNase Ti molecule and absent , T., unpublished data. 132 T. UCHIDA and RNase T2-B) from 500 g. of Taka-Diastase powder. REFERENCES 3. The purified RNase T2 was demon (1) Sato, K., and Egami, F., J. Biochem., 44, 753 strated to be homogeneous as a protein in (1957) chromatography on DEAE-cellulose, gel filtra (2) Sato-Asano, K., J. Biochem., 46, 31 (1959) tion on Sephadex G-75, paper electrophoresis, (3) Naoi-Tada, M., Sato-Asano, K., and Egami, F., sedimentation and N-terminal amino acid J. Biochem., 46, 757 (1959) (4) Egami, F., Kagaku no Ryoiki (in Japanese), 12, analysis. Furthermore, this preparation was 9 (1958) obtained free from various contaminating (5) Takahashi, K., J. Biochem., 49, 1 (1961) enzymes in Taka-Diastase. (6) Koerner, J.F., and Sinsheimer, R.L., J. Biol. 4. Molecular weight of RNase T2 was Chem., 228, 1039 (1957) estimated to be 36,000 from sedimentation (7) Ui, N., and Tarutani, O., J. Biochem., 49, 9 (1961) equilibrium analysis and minimum molecular (8) Sanger, F., Biochem. J., 39, 507 (1945) weight calculated from the analytical results (9) Hirs, C.H.W., J. Biol. Chem., 219, 611 (1956) of amino acid composition. (10) Goodwin, T.W., and Morton, R.A., Biochem.J., 40, 628 (1946) 5. The isoelectric point was found to be around pH 5. (11) Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A., and Smith, F., Anal. Chem., 28, 350 (1956) 6. The N-terminal amino acid was (12) Rushizky, G.W., and Sober, H.A., J. Biol. Chem., determined as glutamine or glutamic acid. 238, 371 (1963) 7. The amino acid composition of RNase (13) Takahashi, K., J. Biochem., 51, 95 (1962) T2 was determined. This enzyme contains (14) Egami, F., Takahashi, K., and Uchida, T,, 6 residues of histidine, 7 residues of tryptophan " Progress in Nucleic Acid Research & Molecular and one residue of methionine. Biology", ed. by J.N. Davidson and W.E. Cohn, 8. Some neutral sugars were contained in Academic Press Inc., New York, Vol. III, p. 59 the purified RNase T2. (1964) 9. Some enzymatic properties of RNase (15) Crestfeld, A.M., Smith, K.C., and Allen, F.W., J. Biol. Chem., 216, 185 (1955) T2, such as stability, pH optimum and (16) Uchida, T., J. Biochem., 57, 547 (1965) sensitivities toward inhibitors and activators, (17) Uchida, T., and Egami, F., J. Biochem., in were investigated in detail. press 10. RNase T2 was separated into RNase (18) Tsugita, A., and Akabori, S., J. Biochem., 46, T2-A and RNase T2-B by the DEAE-cellulose 695 (1959) column chromatography. RNase T2-A was (19) Plummer, T.H. Jr., and Hirs, C.H.W., J. Biol. indistinguishable from RNase T2-B in the Chem., 239, 2530 (1964) enzymatic properties and the properties as a (20) Sato, S., Uchida, T., and Egami, F., Arch. Biochem. Biophys., in press protein. The difference between the both (21) Uchida, T., and Egami, F., " Procedures in fractions was observed only in their carbo Nucleic Acid Research ", ed. by Cantoni, G.L. hydrate components. and Davies, D.R., Harper & Row, Publishers, New York, p. 46 (1966) This work has been supported by a grant to Prof. (22) Takahashi, K., J. Biochem., 52, 72 (1962) F. Egami from the Toyo Rayon Science Foundation, to which the author's thanks are due. The present author wishes to express her sincere thanks to Prof. F. Egami for his guidance and encouragement during this work.