Ribonuclease preparation for the base analysis of polyribonucleotides

.\NAT,YTIC4L

17, 135-142

BIOCHEMISTRY

Ribonuclease

(1966)

Preparation

for the

Base

Analysis

of Polyribonucleotides MICHIKO

HIRAMARU, Department

TSUNEKO

of Biophysics The University

UCHIDA,

and Biochemistry, of Tokyo, Hongo,

AK;D

Faculty Tokyo

FUJI0

EGA>11

of Science.

RNase T, (EC 2.7.7.17, ribonucleate nucleotido-2’-transferase (cyclizing)) isolated from takadiastase has no absolute base specificity and hydrolyzes RNA practically completely to 3’-nucleotides (1). Rushizky and Sober suggested that it, may be useful for the base analysis of RNA (21. Nirenberg et. ‘al. applied it to the analysis of certain oligoribonucleotides (3). RNase T, digestion is certainly more advantageous t,han alkaline hydrolysis, which gives rise to the mixture of 2’- and 3’-nucleotides and may be accompanied by a slight degradation of nucleotide bases, especially of certain minor components. However RNase T, is not. yet available commercially and the purification procedure (4‘1 fro111 takadiastase is fairly laborious. In this paper, therefore, an :Ilternativcl clnzyme preparation will be presented. We will propose that heat-treated takadiastase replace RNare T, fol, the base analysis of polyribonucleotides. It will be shown, that cnzymcxs probably disturbing the analysis are inactivated by the heat trentnlcnt.. \vbilc RNase T, and RNase T, remain sufficiently Rrtiw. MATERIALS

ASD

METHOD,’

Takwdiastase powder .4 was kindly provided by Sankyo (>o., T,ttl.. Tokyo. Low-molecular RNA was the commercial product 1JWjEil’W~ from 2’0~~~1~1 yeast (Toyo Spinning Co.. Ltd., OFaka‘l :md W&F URCC~after purification by the phenol method (51. sRNA was kindly provided by Dr. AI. Tada. This material was prepared from Todu (Toyo Spinning Co.. T,ttl.,i :lncl was frock from trrlninnl :Imino acids. 2’(3’)-Adenylic acid was purchased from Sigma Chemical Co., Lttl. p-Nitrophenyl phosphate and bis-p-nitrophenyl phosphate WI e ~IIYchased from Seikagaku Kogyo Co.. Ltd.. Tokyo. 135

136

HIRAMARU,

UCHIDA,

AND

EGAMI

DEAE-cellulose, a product of Brown Company, Boston, Massachusetts, had a capacity of 0.98 meq/gm. Spectrophotometric measurements were made in silica cells with a l-cm light path in an Ito spectrophotometer (Ito Cho-tampa Co., Ltd., Tokyo). An Hitachi automatic spectrophotometer was used for recording the ultraviolet absorption spectra. Enzyme

Assay

for Phosphatases

The p-nitrophenol method for phosphatase assay (7) was applied as follows. The reaction mixture containing 0.25 ml of 2 mM p-nitrophenyl phosphate (for phosphomonesterase) or of 4 mM bis-p-nitrophenyl phosphate (for phosphodiesterase), 0.25 ml of 0.2 M acetate buffer, pH 4.5, 0.4 ml of distilled water, and 0.1 ml of heat-treated enzyme solution (ca. 17 units), was incubated at 37’C for 18 hr. Then the reaction was stopped by adding 1 ml of 10% TCA. To the resulting solution was added saturated sodium carbonate solution and the optical density at 405 ml-d was measured against a blank, prepared by adding the enzyme solution just before the measurement. Digestion

of RNA

with Heat-Treat’ed

Enzy,me Preparation

The reaction mixture containing RNA (final 10 mg/ml) , acetate buffer, pH 4.5 (final 0.05 M), enzyme solution (8.5 units/mg RNA) and 1 drop of chloroform was incubated at 37°C for 18 hr. Alkaline

Hydrolysis

RNA was dissolved in 0.3 N KOH for 18 hr. Paper Electrophoresis

of RNA

(10 mg/ml)

and Paper Chromatogra.phy

and incubated of Reaction

at 37°C Products

Paper electrophoresis and paper chromatography were carried out on Toyo Roshi 51A papers. The following buffers and solvent systems were used. Bujfer I. Ammonium formate, pH 2.7 (0.063fM formic acid solution was adjusted to pH 2.7 with concentrated ammonia). Bujj’er II. 0.5 M ammonium bicarbonate solution, pH 8. Buffer III. 0.1 M borate buffer, pH 9.2. SoZven,d I. Isopropanol saturated ammonium sulfate aq. solution/O.1 M citrate phosphate buffer, pH 6 (2:79: 19, V/V). Sobvent II. fert-Butanol/ammonium formate, pH 2.7 (l:l, v/v). Column Method

preparation

Chrom.atognaphy

I. DEAE-cellulose column chromatography was carried out according to Uchida (4).

of the enzyme

Method II. DEAE-cellulose column chromatography ot’ alkaline hytlrolyzate from RNA was carried out with a gradient system from 0.01 to 0.09 M ammonium bicarbonate buffer, pH 8.6. :tn(l a ?;tcpwise elution fw 0.45 41 ammonium bicarbonate buffer, pH 8.6. JIefhod III. DEAE-cellulose column chromatography of RNA digest, l)y heat-treated enzyme was carried out wit,h a stepmise clution of 0.01, 0.06. and 0.4F, ill ammonium bicarbonate huffrr. pH 8.6. 1’SPERIMEKTAL

=\ND RESI!J,TS

I. Preparation and Properties of Heat-Twated Scheme 1. Preparation ‘l‘Al
r------I I
of heat-treated

Enzyme

enzynics

powder A (1 gm) Extracted twice with water (2.5 ml, :ultl centrifuged at 3000 rpm for 10 min Slqxrnatant 1 pH adjusted to 1.5 with 1 X’ HCI and centrifrlged at 15,000 rpm for 40 min

r---Prrcaipitatr

I

Snptlrnatant 2 heat-treated at 85°C for 10 min and centrifuged at, 15,000 rpm for 30 min

Precipitate

I

Supernatant 3 dialyzed against, tap water overnight and lyophilized HEAT-TREATED ENZYME PREP.~R,\TJOS (yield ca. 3000 unit&

a Enzyme activity was estimated as usual (8) by measuring t,he optical density at, 260 mp of acid-soluble digestion products from RNA. One unit is defined as an increase in t,he optical density at 260 rnF of 1.0 under the usual assay conditions at pH 4..5.

Heat-treated takadiastase has been prepared as in Scheme 1. The enzyme preparation was found to have 70% RNase activity at pH 4.5 (optimum pH for RNase T,) and 80% RNase activity at pH 7.5 (optimum pH for RNase T,) of the enzyme preparation before heat treatment (supernatant 2). The ratio, amount of RNase Tz/amount of RNase T,, in the enzyme preparation was estimated by separating both enzymes by DEAE-cellulose column chromatography according to Uchida 14). The chromatogram showed that RNaec T, ~a!: responsible for nhout 16% of RNase activity at pH 4.5. TJ. Absence of ihzymes lhturbing

Ba.seAnnlysG

(a) Abscncc of Decrmimse Scting on .Z’- and S’-Nuckotides. According to Minato et al. f6), in takadiastase there is a nonspecific adenoaine

138

HIRAMARU,

UCHIDA,

AND

EGAMI

deaminase which also splits adenylic acid. The absence of such an enzyme in the heat-treated preparation was checked as follows. A reaction mixture containing 2 mg of 2’(3’) -adenylic acid dissolved in 0.1 ml water, 0.05 ml of heat-treated enzyme solution (17 units) and 0.05 ml of 0.2 M acetate buffer, pH 4.5, was kept for 18 hr at 37°C. Then the solution was chromatographed on a paper with solvent system I. On the paper chromatogram 2’- and 3’-adenylic acids were detected but no deamination products. (b) Absence of Phosphodksterase. The heat-treated enzyme preparation was tested for phosphodiesterase activity with bis-p-nitrophenyl phosphate. Since upon measuring the optical density at 405 mp against a blank no release of p-nitrophenol by the enzyme was observed, the enzyme preparation may be regarded as free from nonspecific phosphodiesterase. (c)

Absence

of Phosph80monoestera.se

Disturbing

the Base Analysis.

The heat-treated enzyme preparation releases a small amount of p-nitrophenol from p-nitrophenyl phosphate. Therefore phosphomonoesterase acting on p-nitrophenyl phosphate was not completely inactivated upon being held at 85’C for 10 min. But heat treatment at a higher temperature is not desirable for RNase activity, especially for RNase T,. In order to see if the phosphomonoesterase activity remaining in the heat-treated enzyme preparation disturbs the base analysis of RNA, paper electrophoresis was carried out using buffer I, pH 2.7, for 16 hr at 400 V; 2 mg of RNA was digested with the heat-treated enzyme (17 units) and applied on a paper. At this pH, nucleotides move to the anode and nucleosides move to the cathode. As no nucleoside was detected on the paper, it may be assumed that the heat-treated enzyme preparation is practically free from phosphomonoesterase that disturbs the base analysis of RNA. (d) Absence of Nucleases Producing 5’-Nuckotides. The activity of the enzyme which splits the internucleotide bonds producing 5’-nucleotides was checked with paper electrophoresis using buffer II, pH 9.2; 500 pg of RNA was digested with the heat-treated enzyme (4 units) and applied on a paper. The electrophoresis took 5 hr at 400 V. On the paper 2’- and 3’-nucleotides were found and 5’-nucleotides were not found; that is, heat-treated enzyme preparation is free from nuclease activity producing 5’-nucleotides. III, Amounts

of Heclt-Treated Enzyme Prepa.ration, Needed for Complete Digestion of RNA

In order to know the amount of heat-treated enzyme preparation needed for complete digestion of RNA, a series of the reaction mixtures with different enzyme concentrations was incubated for 18 hr at 37°C. The reaction mixture (2 ml) contained 10 mg of RNA, heat-treated

enzyme solution, and acetate buffer, pH 4.5 (final 0.05 n/l). In parallel with the enzymic digestion, alkaline hydrolysis was carried out using the same RNA solution. The optical density at 260 mp of the acid-soluble fraction of the reaction mixture was measured. Per cent degradation was calculated against optical density at 260 rnp of the alkaline hydrolyzat,es of RNA used. The relation between the amounts of heat-treated enzyme and per cent degradation is shown in Figure 1. As seen in the figure. 50 units of the heat-treated enzyme preparation is enough for 10 mg of RNA to be completely digested and, when completely digested, apparent degradation of RNA with the enzyme is higher by 2% than alkalincb

FIG. 1. Relation between amounts of heat-treated enzyme and per cent degradation. The reaction mixture contained 10 mg RNA, heat-treated enzyme solution, and acetate buffer, pH 4.5 (final 0.05 MJ. Per cent degradation was calculated as described in the text.

degradation; 8.5 units of the heat-treated RNA to be completely digested.

enzyme is enough for 1 mg of

(a) Absence of Xucleoside B’,S-Cyclic Phosphates. In order to see ii the intermediates of the enzymic digestion, nucleoside 2’,3’-cyclic phosphates, were present in the RNA digest, a mapping was carried out as follows: 1 mg of RNA was digested with 8.5 units of heat-treated enzyme and applied on a paper; electrophoresis was carried out at pH 8 using buffer II for 3 hr at 400 V followed by paper chromatography with solvent system I. On the map, nucleoside 2’,3’-cyclic phosphates were not detected. (b) Alkaline Hydrolysis Resistant Fraction. The presence of the components resistant to alkaline hydrolysis was reported by Smith and Dunn (8) and it was confirmed as follows: 20 mg of RNA was dissolved in 2 ml of 0.3 N KOH and incubated for 18 hr at 37°C. After neutralization with Perchloric Acid, the hydrolyzate was loaded on a DEAE-cellulose column (1 X 50 cm) and chromatography method II was carried out.

140

HIRAMARU,

UCHIDA,

AND

EGAMI

From the chromatography it was found that all the mononucleotides were eluted at salt concentration below 0.06 M and a remaining small fraction was eluted at higher salt concentration. This fraction may be called alkaline hydrolysis core. The core was found to be 2.5%. (c) He;&-Tre’ated Enzyme Resistant Fraction in RNA. The mixtures of the enzyme solution and acetate buffer, pH 4.5, with and without RNA ).Ol M

0.06

M

045

M

0.45

M

--

-I-

100

60

I

0.01 M - -I+--

0.06

M --

c

(“’,00 I

400 Effluent

800 volume

(ml)

FIG. 2. Elution patterns of ensymic digest of RNA (a) and the heat-treated enzyme preparation (b) on DEAE-cellulose columns (1 x 30 cm). Loud: (a) enzymic digest from 10 mg of RNA, (b) incubated mixture of the enzyme solution and acetate buffer, pH 4.5. El&on: stepwise, of 0.01, 0.06, and 0.45M ammonium bicarbonate buffer, pH 8.6. Elution curves were automatically recorded at 254 rnw by LKB-Wicord ultraviolet absorption.

were incubated for 18 hr at 37°C and DEAE-cellulose column chromatography met’hod III was carried out. The fractions eluted at each salt concentration were collected and the optical density at 260 mp was measured. The heat-treated enzyme core calculated as the difference of the total optical density of the fractions with RNA (Fig. 2a) and without RNA (Fig. 2b) added was found to be 3.5% and higher by 1% than the alkaline hydrolysis core.

RNASE

PREPARATION

FOR

RNA

ANALYSIS

141

On the other hand, in order to estimate. not as the difference, but directly, the amount of the core, we tried to rcmo\e ultraviolet-absorbing substances coming from the enzyme preparation. :\n enzyme solution incubated with acetate buffer, pH 4.5, for 18 ln WE treated with 9Qy phenol and DEAE-cellulose column cllrolllatogr:II)lly method III wai carried out. Fro111 the chromatography, ultrnr~iolet-al)~ol,lJillg substances were considered to be removed by the phenol treatment. Then, the RNA digest with the heat-treated enzyme was treated with 90% phenol an(l column chromatography III was carried out. The chromatogram showed that the heat’-treat,ed enzyme core was 3.4Ji: . in good agreement with tlrca result mentioned above. .4s the loss of digests by the phenol treatment is considerable, it might be well to carry out the :~n:tlysis without. the phenol treatment lvhen onl? very small amounts of polyribonucleotides are available. (d) Jlapping of sRA’-1 Digest. The heat-treated enzyme preparation was shown to produce minor nucleotides along with four major CO~IIponentl: by sRNA tligestion. Mapping of sRN-1 was carried out, a:: described by Miurw (10) : 2 mg of sRNA \\-a~ digested with the heattreated enzyme (17 units) and applied on a What’man 3MM paper. Electrophorcsis took 16 hr at 600 V followed by ascending chromatopraphp with solvent system II. The spots W’I’C identified by their absorljtion spectra and by comparing with the map of IiNnsc T, digest I)!. ITchida (11). DISCUSSION

Heat-treated takadiastase powder A was found to qjlit RNA pramtically completely to 3’-mononucleotides and to be practically free from enzymes such as phosphatases and deaminase. Therefore it) map be used in most casesfor the base analysis of RNA in plncc of purified RNaae T,. The nature of the fraction resistant to heat-treated takadiastase in RNA remains to be elucidated. It may contain, among others, 2’-omethylated nucleotides and, if any, tr-ribosidc derivatives (12‘1. It is remarkable that the apparent amount of the resistant fraction i,. rbven lower t,han that of alkaline hydrolysis so far measured by the increase of t,otal opt,ical density. It might hr due to the slight dcgrndation of nncleotide basesin alkaline hydrolysis. Heat t,rcatmcnt for 10 min at 85°C was selected because treatment for 10 min at 80°C was insufficient, to inactivate l~hosphomonoorterasc nntl treatment for 10 min at 90°C decreased too much the RNase nrtiritv at pH 4.75. This heat,-treated takadiastase preparat,ion may he u.-;ed in place 01 RNasr ‘T1 not only far base ana.lvsis but also in other npplications ruclr

142

HIRAMARU,

UCHIDA,

ASD

as the specific cleavage of nucleoside 2’,3’-cyclic 3’-phosphates.

EGAMI

phosphates to nucleoside

SUMMARY-

Takadiastase extract heated at 85°C for 10 min at pH 1.5 may be used in place of RNase T, as an RNase preparation for the base analysis of oligo- and polyribonucleotides. REFERENCES 1. UCHIDA, T., AND EGAMI. F., J. Japan Biochem. Sot. 34,407 (1962). 2. RUSHIZKY, G. W., AND SOBER, H. A., J. Biol. Chem. 238, 371 (1963). 3. NIRENBERGI, M., LEDER, P., TRWIN, J., ROTTMAN, F., AND O’NEAL, C., Proc. Natl. Acud. Sot. U. S. 53, 1161 (1965). 4. UCXIDA, T., AND EGAMI, F., in “Procedures in Nucleic Acid Research” (G. L. Cantoni and D. R. Davies, eds.), p. 7. Harper and Row, New York, 1966. 5. KIRBY, K. S., Biochem. J. 64, 405 (1950). 6. MINATO, S., TAGAWA, T., AND NAKANISHI, K., J. Biochem. (Tokyo) 58, 519 (1965). 7. AKAMATSU, S., in “Methods in Enzymology” (“Koso Kenkyuho” in Japanese) (S. Akabori, ed.), Vol. 2, p. 45. Asakura Shoten, Tokyo, 1956. 8. EGAMI, F., TAKAHASHI, K., AND UCHIDA, T., in “Progress in Nucleic Acid Research and Molecular Biology” (J. N. Davidson and W. E. Cohn, eds.), Vol. 3, p. 59. Academic Press, New York, 1964. 9. SMITH, J. P., AND DUNN, D. B., Biochim. Biophys. Acta 31, 573 (1959). 10. MIURA, K., J. Mol. Biol. 8, 371 (1964). 11. UCHID.4, T., J. Biochem. (Tokyo), to be published; Information Exchange Group No. 7. #457. (1966). 12. GASSEN, H. G., AND WITZEL, H., Biochim. Biophys. Acta 95, 244 (1965).