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Ribonuclease from Streptomyces erythreus: Purification and properties
636
BIOCIIIMICAET BIOPHYSICAACTA
BB& 96713
R I B O N U C L E A S E FROM S T R E P T O M Y C E S
ERYTHREUS
P U R I F I C A T I O N AND P R O P E R T I E S
NOBUO YOSHIDA, HIDEO INOUE, ATSUSHI SASAKI AND HIDEO OTSUKA Shionogi Research Laboratory, Shionogi and Co., Ltd., Fukushima-ku, Osaka (Japan) (Received September 7th, I97o)
SUMMARY
Ribonuclease from Streptomyces erythreus has been purified b y sequential chrom a t o g r a p h y with DEAE-cellulose, DEAE-Sephadex and QAE-Sephadex. The purified enzyme gives a simple band on polyacrylamide disc electrophoresis and an examination b y sedimentation velocity indicates a high degree of homogeneity for the preparation. The purified ribonuclease has a sedimentation coefficient of 1.61 S, and a molecular weight of I I 9o0 as determined by a sedimentation equilibrium experiment is consistent with those estimated b y gel filtration on Sephadex G-75 and from its amino acid composition. The isoelectric point was found to be around p H 4.5. The amino acid composition of the ribonuclease was determined. I t shows a high proline content and contains two residues of half cystine corresponding to a single disulfide bond. Neither t r y p t o p h a n nor methionine was found in the enzyme.
I NTRODUCTION Ribonuclease is produced b y several microorgal~isms as an extracellular and/or a n intracellular enzyme. In a previous study carried out b y TANAKA1, a ribonuclease was isolated from Streptomyces erythreus and exhibited a substrate specificity similar to ribonuclease T 1 which catalyzed the splitting of phosphodiester bonds of 3'-guanylic acid in RNA and the 2'-ester bond of guanosine 2',3'-cyclic phosphate. In addition, a further demonstration concerning the specificity of the former has been reported using yeast low molecular weight RNA as a substrate 2. An interest in the comparative biochemistry, particularly the protein structure, of ribonucleases which show the same substrate specificity prompted us to undertake the purification of a ribonuclease from S. erythreus, since the lack of a pure preparation of the ribonuclease has not permitted studies of its substantial properties and kinetics. The present report is concerned with studies on the purification and characterization of the physical and chemical properties of ribonuclease from S. erythreus. Some discussion will be concerned with a comparison of the amino acid composition of the ribonuclease with those of ribonuclease T 1 and bovine pancreatic ribonuclease (ribonuclease A).
Biochim. Biophys. Acta, 228 (1971) 636-647
PURIFICATION AND PROPERTIES OF RIBONUCLEASE S t
637
MATERIALS AND METHODS
DEAE-cellulose (o.82 mequiv./g) was purchased from Brown Co. and DEAES~phadex, QAE-Sephadex and Sephadex G-75 were obtained from Pharmacia, and yeast RNA used for ribonuclease assay was purchased from Kokoku Rayon and Pulp Co. (Tokyo).
Ribonuelease assay The enzymatic activity of ribonuclease was determined by measuring the absorbance at 260 m# of the acid-soluble digestion products from yeast RNA according to the method described by TANAKA3 using Tris-HC1 buffer instead of phosphate, and the specific activity (enzyme unit) was calculated in accordance with the definition presented previously by TANAKA3.
Electrophoretic analyses Polyacrylamide gel electrophoresis was performed in a Canalco analytical disc electrophoresis apparatus at room temperature with a 15 % cross-linked gel according to the procedure recommended by ORNSTEIN4 and DAVIS5. Gels were stacked at pH 7.5 with samples containing 200/~g of enzyme dissolved in 40 % sucrose tray buffer solution (pH 7.0) and were subjected to electrophoresis at 2.0 mA per gel. Gels were stained with 1 % amido black dissolved in 7 % acetic acid for I h and were electrophoretically de-stained (8 mA per gel) in 7 % acetic acid.
Ultracentri]ugal analysis A Hitachi model UCA-I ultracentrifuge was used to measure the sedimentation constant and to determine the molecular weight by sedimentation equilibrium. All analyses were performed in o.I M Tris-HC1 buffer, pH 7-4. Sedimentation velocity experiments were performed at 51 900 rev./min in a synthetic boundary cell. The sedimentation constant was corrected to a value corresponding to water at 20 °. The sedimentation equilibrium procedure was done with a double sector centerpiece at 20 350 rev./min. Schlieren images and Rayleigh interference fringes were recorded on Kodak metallographic plates, a n d the positions of the patterns were analyzed in a microcomparator (Shimadzu, Model SR-2). The partial specific volume of the protein was calculated from its amino acid composition by the method of COHN AND EDSALL e.
Amino acid analyses A sample of enzyme (approx. 2 mg) was suspended in I ml of glass-distilled HC1, de-aerated and sealed, and hydrolyzed at 11o% Immediately prior to analysis, hydrolyzates were evaporated to dryness under reduced pressure at 55 ° in a rotary evaporator. Amino acid analyses were performed in a Jeol's analyser model JLC-3BC (Japan Electron Optics Lab. Co., Tokyo) with optimal precision in the 0.05 #mole range. Separate samples were oxidized with performic acid for the determination of cysteic acid by the procedure of HIRS~. Tryptophan was estimated spectrophotometrically in an unhydrolyzed sample by the method of GOODWlN AND MORTONs and the method of SPIES and CHAMBERS~'1° using p-dimethylaminobenzaldehyde. Biochim. Biophys. Acta, 228 (1971) 636--647
~. YOSHIDAet al.
638
Molecular 7e,eight determination by gel/iltration The method of WHITAKER11 was applied to the purified ribonuelease. A column (1.6 cm × 88 cm) of Sephadex G-75 was equilibrated at room temperature with a buffer, pH 6.o, composed of o.I M sodium acetate and o.I M NaG1 (J = o.194 ). To measure the void volume (V0) of the column, blue dextran 2ooo was used. Other markers were bovine serum albumin (tool. wt. 7 ° ooo), pepsin (tool. wt. 35 ooo), ~-chymotrypsin (mol. wt. 25 ooo), bovine pancreatic ribonuelease (mol. wt. 14 ooo). 2-ml fractions were collected and monitored at 28o m/t, and the flow rate was regulated to 3o ml/h.
RESULTS
Purification procedure Step I and Step II (Table I) were performed according to TANAKA'S1 method and further steps (Steps I I I , IV and V) were followed by sequential chromatography at 4 ° . TABLE I SUMMARY OF PURIFICATION OF RIBONUCLEASE FROM S.
Step and treatment
Starting broth Acrinol p r e c i p i t a t e Batchwise DEAE-cellulose treatment III. DEAE-cellulose chromatography IV. D E A E - S e p h a d e x (A-25) chromatography V. Q A E - S e p h a d e x (A-5o ~ chromatography I. II.
Total protein
erythreus .Specific activity (units/A 280 mu)
Puri[ication factor
Yield (%)
IOO 1 13o g 2o.8 g
3° 270 12oo
9 4°
ioo 4° 29
55 ° m g 142 m g
125oo 45000
42o I5oo
io 6.9
lO6 m g
52000
i83o
6.o
Step I I I chromatography on DEAE-cellulose. The fraction treated batchwise with DEAE-cellulose was purified by DEAE-cellulose chromatography, the column being developed with o.i M acetate buffer, pH 4.0, containing 0.03 M NaC1 until an active fragment was eluted (Fig. I). The active effluents were pooled and the enzyme was subsequently concentrated by the addition of (NH4)2SO a to 66 °,o saturation. The precipitate produced during one day was dissolved in a minimal volume of distilled water, dialyzed against distilled water and lyophilized. Step I V chromatography on DEAE-Sephadex. The crude enzyme obtained in Step III, 0.35 g, was dissolved in 35 ml of o.i M Tris-HC1 buffer containing o.I M NaCI at pH 8.o and placed on a DEAE-Sephadex (A-25) column, 2 . 6 c m × 3 o cm, equilibrated with the same buffer. An exponential gradient elution was carried out from o.I M NaC1 in the buffer to the buffer containing 0.3 M NaC1 with a 1.2-! mixing chamber. Fractions of 15 ml were collected at a flow rate of 60 ml/h. Fig. 2 shows the result of the gradient elution portion of this column step. Tile last protein peak eluted with this gradient system contained the ribonuclease activity. Fractions conBiochim. Biophys. Acta, 228 (1971) 636-647
639
PURIFICATION AND PROPERTIES OF RIBONUCLEASE S t
? 6
• I.0 I
> tJ
::L E o oo o4
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o
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.-o-
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o2
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QI
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3ooo
2oo0 Effluent ml
Fig. I. Chromatography of a batchwise DEAE-cellulose-treated fraction on a column, 2.6 cm × 50 cm, of DEAE-cellulose. A sample of 4 g in 4 ° ml of o.i M acetate, pH 4.o, was applied to the columt~. Effluent fractions of 18 nil were collected at a rate of i5o ml]h.
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1 0.8
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i500"
~000
o
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l~ig. 2. Chromatography of partially purified enzyme on DEAE-Sephadex (A-25). Column: 2.6 cm × 3 ° cm. Load: 35 ° mg of DEAE-cellulose chromatographically purified enzyme. Elution: exponential gradient from o.i M NaC1 in o.I M Tris-HCl buffer (pH 8.o) to o.3o M NaC1 in the buffer ~ i t h a 12oo m] mixing chamber, at the flow rate of 60 ml/h, 15 ml per fraction.
t a i n i n g t h e e n z y m a t i c a c t i v i t y w e r e p o o l e d , a d j u c t e d t o p H 4.0 a n d c o n c e n t r a t e d b y t h e a d d i t i o n of ( N H 4 ) 2 S 0 4 as i n S t e p I I I . A f t e r d i a l y s i s a n d l y o p h i l i z a t i o n , a p p r o x . I i O m g of t h e p r o t e i n w e r e o b t a i n e d , w i t h a s p e c i f i c a c t i v i t y of 45 ooo u n i t s . Step V chromatography on QA E-Sephadex. 255 m g of t h e e n z y m e p r e p a r a t i o n o b t a i n e d f r o m t h e p r e c e d i n g c h r o m a t o g r a p h y w a s d i s s o l v e d i n 25 m l of o.~ M T r i s HC1 b u f f e r , p H 7.4, plus o . I o M NaC1 a n d w a s c h r o m a t o g r a p h e d o n a Q A E - S e p h a d e x (A-5o) c o l u m n , 2.6 c m × 52 c m , e q u i l i b r a t e d w i t h t h e b u f f e r c o n t a i n i n g o . I o M NaC1. A g r a d i e n t e l u t i o n s y s t e m f r o m o . I o t o 0.27 M NaC1 w i t h a I-1 m i x i n g c h a m b e r w a s c a r r i e d o u t b y t h e s a m e m e t h o d a s i n S t e p I V . F r a c t i o n s of 15 m l w e r e c o l l e c t e d a t a
Biochim. Biophys. Acta, 228 (1971) 636-647
64o
~
N. YOSHIDAet al.
2.0 t3 *t
~'1.5 0 CO
c~ 2.0 x
Lo
° "=:L
.~0.5
O4
a4 ~ 500
1000
1500
2000
Effluent ml
o ID <1
Fig. 3. Final purification of ribonuclease from S. erythreus on Q A E - S e p h a d e x (A-5o). Column: 2.6 cm × 52 cm. Load: 255 m g of D E A E - S e p h a d e x c h r o m a t o g r a p h i c a l l y purified enzyme. Elution: exponential gradient f r o m o. I iV[ NaC1 in o. I M Tris-HC1 buffer, p H 7.4, to o.27 M NaC1 in the buffer w i t h a i-1 mixing chamber, a t the frow r a t e of 18 ml/h, 15 ml per fraction.
flow rate of approx. 18 ml/h. As shown in Fig. 3, the enzyme active fraction yielded a symmetrical peak (about 200 mg of lyophilized protein). Upon re-chromatography of the purified enzyme thus obtained, on QAESephadex with a one-step elution with o.17 M NaC1 in o.I M Tris-HC1 buffer (Fig. 4), or with the same gradient elution as described above (Step V), the enzyme was eluted as a single symmetrical peak with the respect to the enzymatic activity as well as to the protein. The specific activity was increased about i8oo-fold by the total purification for the five steps, i.e. from 5 ° ooo to 55 ooo units. Table I summarizes the results obtained when IOO 1 of starting broth were processed b y the purification procedure just outlined. The over-all yield of enzymatic activity in the described experiment was about 6 °/0 and approx. IOO mg of the purified protein was prepared.
/ o.31o |/
6°r 5.51-
0 O2L__j
~
~
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o.~
40O Effluent ml Fig. 4. Re-chromatography of final purified preparation, Step V, on QAE-Sephadex A-5o. Column: 1.6 c m × 5 4 cm. Load: 36.5 mg of QAE-Sephadex chromatographically purified enzyme. Elution: o.I M T r i s - H C l buffer containing o.i 7 M NaCI, p H 8.0, at the flow rate of 5 ml/h, ,-o ml per fraction.
Biochim. Biophys. Acta, 228 (1971) 636-647
PURIFICATION AND PROPERTIES OF RIBONUCLEASE S t
641
Criteria o/purity Disc electrophoretic patterns. The disc electrophoretic patterns shown in Fig 5 represent the protein bands in the sequential fractions during purification, and the final product shows a single band as shown in Fig. 5c.
Fig. 5. Polyacrylamide gel electrophoretic patterns of fraction obtained during the isolation of ribonuclease from S. ervthreus. 15 % small-pore gel, running at p H 7.0, 2.0 mA per gel for i h, 20o/tg of protein. A.The active fraction from DEAE-cellulose chromatography step. 13. The active fraction from DEAE-Sephadex chromatography step. C. The final product after QAE-Sephadex chromatography step.
Ultracentri/ugal pattern. Fig. 6 shows the ultracentrifugal Schlieren pattern of a solution of purified enzyme in o.I M Tris-HC1 buffer, pH 7.4, at a protein concentration of 5.2 mg/ml. The enzyme showed a single symmetrical peak with a sedimentation coefficient of 1.61 S. In a separate experiment carried out at a protein concentration of 3.3 mg/ml, the same s20,w value was obtained.
Fig. 6. Ultracentrifuge patterns for sedimentation velocity experiments of ribonuclease from S. erythreus in a synthetic boundary cell at 51 90o rev./min and 21.3 °. The protein concentration is 5.z mg/ml in o.i M Tris-HCl buffer, p H 7.4. The pictures were taken at i (A), 30 (B) and 60 (C) min after reaching the final speed.
Molecular weight Gel /iltration method. The purified enzyme was passed through a Sephadex G-75 column, and the elution pattern was compared with several markers which Biochim. Biophys. Acta, 228 (1971) 636-647
N. YOSHIDA et al.
642
Ve/Vo ! 2 . 0 ~ I. 6
I"I 4.0
"~hymolrypsi n
I
I I I IBovineIum Sre 4.2 4.4 4.6 Logorithrnof Molecul¢,rWeight
~
4.8
i
Fig. 7. Relationship b e t w e e n Ve/Vo values and molecular weights of proteins used in determining the molecular weight of ribonuclease from S. erythreus.
showed a linear relation between Ve/Vo (Ve; elution volume of each marker; Vo, void volume) and the logarithms of the molecular weights for the reference proteins (Fig. 7). On the basis of this relationship and the Ve/Vo value of 2.08 obtained for the enzyme, a molecular weight, of approx. 12 ooo was found for the ribonuclease of S. erythreus. This molecular weight value is supported by the experimental result that the enzyme is dialyzable through a collodion bag (Sartorius Membranfilter) under reduced pressure while it is not dialyzable on a cellulose tubing (Visking). TABLE II AMINO ACID COMPOSITION OF HYDROLYZATE OF RIBONUCLEASE FROM S. erythreus
Residue
Lys His Arg Asp
Thr Set Glu Pro Gly Ala Cys Val Ile Leu Tyr Phe 1NH~
Total
Residues per
N (% of
zoo g o/ protein
total N)
Calc. residues /or tool. wt. 3:2000
2.27 2-43 12.9o 12.oo 6.81 2.27 16.6o 6.05 6.67 3.82 1.37 4 .68 2.72 4.98 I 1.6o 6.15 o.16
3.05 4.57 28.4 8.83 5.80 2.25 I 1.04 5.37 10.o6 4.62 1.15 4 .o6 2.07 3.78 6.12 3.60 3.28
2.13 2-13 6.o7 12.4 8.1o 3.14 I5. 5 7.49 14- I 6.39 1.6o 5.74 2.88 5.28 8.54 5.03 7.5
lO2.51
*
IO8.O 5
* E x c l u d i n g N H a. Biochim. Biophys. Acta, 228 (1971) 636-647
lO6.52"
Nearest intregral number o~ residues per molecule 2 2 6 12 8 3 16 8 14 6 2 6 3 5 9 5 lO7
643
PURIFICATION AND PROPERTIES OF RIBONUCLEASE S t
Amino acid composition. The amino acid composition of the ribonuclease is shown in Table II, and the molecular weight was calculated to be I I 921. Sedimentation equilibrium method. The molecular weight of the ribonuclease was estimated independently from its sedimentation behavior. The protein was analyzed by the sedimentation equilibrium method at several concentrations (I.4I, 1.175 and 0.82 mg/ml). The equilibrium concentration of the protein at the meniscus was determined according to LA BAR'S 12procedure. Semi-logarithmic plots of fringe displacement against the square of the centrifugal radius afforded straight lines: a typical plot is presented in Fig. 8. This linear relationship as well as the symmetric
I
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I 51.0
T.40
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o
T.oo
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I 48.0
I 49.0 r 2 (Gin a )
Fig. 8. S e d i m e n t a t i o n e q u i l i b r i u m n l t r a c e n t r i f u g a t i o n of ribonucIease f r o m S. erythreus of t h e initial c o n c e n t r a t i o n 1.175 m g / m l a t 2o 35o r e v . ] m i n a t 29.2 °. Plot of l o g a r i t h m of t h e a b s o l u t e fringe d i s p l a c e m e n t , l o g / , as a f u n c t i o n of t h e s q u a r e of radial d i s t a n c e f r o m t h e c e n t e r of r o t a t i o n , $,2.
boundary shape in the sedimentation velocity patterns supports the homogeneity of the ribonuclease in molecular weight. Using a partial specific volume of o.711 ml/g, calculated from the amino acid composition, molecular weights under these conditions computed from the data obtained from three different concentrations were almost independent of the enzyme concentration, giving I I 9005100. The results of these three independent methods are in good agreement, indicating a molecular weight, of approx. I I 900 for the ribonuclease of S. erythreus.
Isoelectric point To estimate the isoelectric point of the ribonuclease, an electrophoresis on cellulose acetate film (OXOID) was carried out. The ionic strength of all buffer solutions was o.I and the pH value ranged from 3.0 to 6.013. After drying and staining with Ponceau 3R (0.4 % aqueous solution containing 3 % trichloroacetic acid), a single stained band was produced, and the distance migrated from the origin was measured on strips carrying all the bands. The electrophoretic mobility of the enzyme as a function of pH is shown in Fig. 9, which represents an isoelectric point near p H 4.5. Biochim. Biophys. Acta, 228 (1971) 6 3 6 - 6 4 7
~. YOSHIDA et al.
644
+L5 0
\
+1.0
cO
+0.5 °~
0 Q O C
-0.5
0
~5
-I .0 !
2.0
I
3.0
I
4.0
|
,5.0
I
6.0
pH
Fig. 9. E l e c t r o p h o r e t i c m o b i l i t y of ribonuclease f r o m S. erythreus. E x p e r i m e n t s were p e r f o r m e d u n d e r c o n d i t i o n s in w h i c h t h e electrophoresis w a s r u n for 45 m i n w i t h 16. 7 V / c m (2.2 m A / c m ) a t 4 °, in either g l y c i n e - H C l b u f f e r ( O ) or a c e t a t e (O) a t a n ionic s t r e n g t h of o.i.
Amino acid composition The mean values of four analyses for 24 and 48 h (duplicate analyses at each hydrolysis time) are represented in Table II. No significant increase of valine and isoleucine was observed even after 72 h of hydrolysis. The numbers for threonine and serine are the values obtained from duplicate analyses of the 8-h hydrolysate. Half-cystine was independently determined as cysteic acid and its value was 1. 9 residues per molecule as compared with 1.6 residues obtained from Table I I on the un-oxidized protein. No methionine nor t r y p t o p h a n could be detected in a n y analysis. The analytical results summarized in Table I I account for lO3 O/,oof the weight of the ribonuclease and the total number of amino acid residues is lO 7. The sugar content in the enzyme was examined by the method of DUBOlS et al. 1~, and found to be nearly zero.
DISCU SSION
The isolation of this enzyme is a necessary step in verifying a correlation between the enzymatic function, particularly binding of substrate to the active site, and the chemical structure as compared with those of ribonuclease T 1. In the present paper, ribonuclease from S. erythreus has been purified b y sequential chromatography with anion-exchange cellulose and Sephadex. The enzyme preparation appears to be homogeneous by the following criteria: re-chromatography on QAE-Sephadex, polyacrylamide disc gel electrophoresis, cellulose acetate film electrophoresis, sedimentation velocity and the linearity of the sedimentation equilibrium plot. For convenience, it appears appropriate to assign a trivial name to the purified ribonuclease, which we propose to call ribonuclease St. The amino acid composition and some physicochemical parameters of the purified enzyme have been determined. A molecular weight of i i 900 has been obtained Biochim. Biophys. Acta, 228 (1971) 636-647
PURIFICATION
AND PROPERTIES
OF RIBONUCLEASE
645
St
by three independent methods, Sephadex G-75 gel filtration, sedimentation equilibrium and amino acid composition. The amino acid composition of ribonuclease St differs notably from those oI ribonuclease T 1 and the bovine pancreatic ribonuclease (ribonuclease A) as shown in Table III. There are several interesting features in Table III. First of all, methionine TABLE
III
AMINO ACID COMPOSITION OF RIBON!dCLEASE St~ T 1 AND A
A m i n o acid
N u m b e r o[ residues per molecule Ribonudease St
Lys His Arg Asp Thr Ser Glu Pro Gly Ala Cy~ Val Met Ile Leu Tyr Phe Trp Total
Ribonuclease T I
Ribonuclease A
2 2 6 12 8 3 16 8 14 6 2 6 o 3 5 9 .5 o
i 3 i 15 6 15 9 4 12 7 4 8 o 2 3 9 4 I
io 4 4 15 io 16 II 4 3 12 8 9 4 3 2 6 3 o
107
105
124
and tryptophan are absent from ribonuclease St so far examined, whereas one tryptophan residue was found in ribonuclease T 1 and 4 methionine residues per ribonuclease A molecule. As to the function of the tryptophan residue of ribonuclease 3"1,it has been recently published that inactivation was caused by N-bromosuccinimide oxidative modification 15 and by the use of 2-hydroxy-5-nitrobenzyl bromide ~6. However, it appears that the tryptophan in ribonuclease T~ has no direct role in the catalytic process, but may be important in maintaining the proper conformation of the active site. From the findings that ribonuclease St has one more phenylalanine residue than ribonuclease 3"1 and that the tryptophan residue is found at the hydrophobic region ( T y r - G l u - T r p - P r o - I l e - L e u ) 17 of ribonuclease T1, a prediction m a y be made that ribonuclease St contains the phenylalanine residue at the position corresponding to the t r y p t o p h a n in the sequence of ribonuclease T r Secondly, it is iloteworthy that among these ribonucleases there are apparently some differences in the amount of several amino acids, such as half-cystine, serine, arginine and proline. In several experiments with native and oxidized ribonuclease St, the half-cystine content of the enzyme was found to be lower than those of ribonuclease T 1 and ribonuclease A. The number of half-cystine residue of ribonuclease St corresponds to a single disulfide bond per molecule, while in ribonuclease T 1 there Biochim. Biophys. Acta, 228 ( 1 9 7 1 ) 6 3 6 - 6 4 7
646
N. YOSHIDAet al.
are two disulfide bonds and there are 4 bonds in pancreatic ribonucleases isolated from bovine, porcine and rat sources TM. It is interesting that animal pancreatic ribonucleases show a more rigid secondary structure, owing to cross-linking b y the four disulfide linkages, compared with those of microbial ribonucleases, and that the two disulfide bridges in ribonuclease TI found in the fungus give a more rigid molecular construction than that of ribonuclease St in the bacterium, Streptomyces. Ribonuclease St exhibits a markedly different amino acid composition from the other ribonucleases; in spite of having the same per cent of threonine residues, the serine content of the enzyme is somewhat lower than that of the others and, in contrast, the arginine content is extremely high. The fact that ribonuclease St, an arginine-rich protein, is isoelectric near p H 4.5 suggests that at higher p H regions than the isolelectric point, the greater part of side chains of Asx and Glx residues in the protein exist not as amidation states nor as salt linkages formed between anionic groups, but as carboxylate ions. Finally, the amino acid composition of ribonuclease St shows a high proline content (7.5 residues per cent) which is twice that of ribonuclease T v The ~-helix coil of a protein m a y be disrupted at the points of the proline residues, and it hasbeen estimated that a protein with 8 °/o proline randomly distributed throughout the polypeptide chain would have an almost complete absence of ~-helix conformation '". Hence ribonuclease St would be expected to have either a low helical content or proline-rich regions of considerable size. On the other hand, it is suggested that ribonuclease T~ has a considerably regular conformation compared with ribonuclease St, since the content of proline residues of the former is approx. 4 %. The helical content of ribonuclease St, in fact, could not be estimated from our preliminary results of the optical rotatory dispersion and circular dichroism of the enzyme (N. YosrnDA, K. KURIYAMA,T. IWATA AND H. OTSUKA, unpublished) while the content of ribonuclease T 1 was calculated to be 20 to 30 % b y the Moffit-Yang method ~°. A more detailed comparison must await the outcome of amino acid sequence studies in progress with ribonuclease St.
ACKNOWLEDGMENTS
The authors wish to t h a n k Dr. K. T a n a k a for his kind advice of the purification procedure.
REFERENCES i K. TANAKA,J. Biochem. Tokyo, 5 ° (1961) 62. 2 K. TANAKA AND G. L. CANTONI,Bichim. Biophys. Acta, 72 (1963) 641. 3 K. TANAKA,in G. L. CANTONI AND D. R. DAVIS, Procedures in Nucleic Acid Research, H a r p e r a n d Row, N e w Y o r k , 1966, p. 14. 4 L. ORNSTEIN, Ann. N. Y. Acad. Sci., 121 (1964) 321. 5 B. J. DAVIS, Ann. N. Y. Acad. Sci., 121 (1964) 4o4 . 6 E. J. COliN AND J. T. EDSALL, Proteins, Amino Acids and Peptides, R e i n h o l d , N e w Y ork, 1943, p. 375. 7 C. H. W. HIRs, J. Biol. Chem., 219 (1956) 611. 8 T. W. G o o n w I N AND R. A. MORTON, Biochem. J., 4 ° (1946) 628. 9 J. R. SPIES AND D. C. CHAMBERS, Anal. Chem., 20 (1948) 3 o. i o J. R. SPIES AND D. C. CHAMBERS, Anal. Chem., 21 (1949) 1249.
Biochim. Biophys. Acta, 228 (1971) 636-647
PURIFICATION AND PROPERTIES OF RIBONUCLEASE S t ii 12 13 14 15 16 17 18 19
20
647
J. R. VtfHITAKER,Anal. Chem., 35 (1963) 195 TM F. E. LA BAR, Proc. Natl. Acad. Sc4. U.S., 54 (1965) 31. G. L. MILLER AND R. H. GOLDER, Arch. Biochem., 29 (195 o) 420. M. DUBOlS, K. A. GILLES, J. K. HAMILTON, P. A. REBERS AND F. SMITH, Anal. Chem., 28 (1956 ) 35 ° • S. KAWASHIMA AND T. ANOo, Int. J. Protein Res., I (1969) 185. K. TAKA~IASHI, J. Biochem. Tokyo, 67 (197 o) 541. K. TAKAHASHI, J . Biol. Chem., 24o (I965) 4117 • V. N. REIr~rtOLD, F. T. DUr~NE, J. C. WRISrON, M. SCHWARZ, L. SARDA AND C. H. W. HIRS, J. Biol. Chem., 243 (1968) 6482. A. G, SZENT-Gy6RGX'I AND C. COHEN, Science, 126 (1958) 697. K. TAKAHASHI, J. Biochem. Tokyo, 60 (1966) 239.
Biochim. Biophys. Acta, 228 (1971) 636-647
BIOCIIIMICAET BIOPHYSICAACTA
BB& 96713
R I B O N U C L E A S E FROM S T R E P T O M Y C E S
ERYTHREUS
P U R I F I C A T I O N AND P R O P E R T I E S
NOBUO YOSHIDA, HIDEO INOUE, ATSUSHI SASAKI AND HIDEO OTSUKA Shionogi Research Laboratory, Shionogi and Co., Ltd., Fukushima-ku, Osaka (Japan) (Received September 7th, I97o)
SUMMARY
Ribonuclease from Streptomyces erythreus has been purified b y sequential chrom a t o g r a p h y with DEAE-cellulose, DEAE-Sephadex and QAE-Sephadex. The purified enzyme gives a simple band on polyacrylamide disc electrophoresis and an examination b y sedimentation velocity indicates a high degree of homogeneity for the preparation. The purified ribonuclease has a sedimentation coefficient of 1.61 S, and a molecular weight of I I 9o0 as determined by a sedimentation equilibrium experiment is consistent with those estimated b y gel filtration on Sephadex G-75 and from its amino acid composition. The isoelectric point was found to be around p H 4.5. The amino acid composition of the ribonuclease was determined. I t shows a high proline content and contains two residues of half cystine corresponding to a single disulfide bond. Neither t r y p t o p h a n nor methionine was found in the enzyme.
I NTRODUCTION Ribonuclease is produced b y several microorgal~isms as an extracellular and/or a n intracellular enzyme. In a previous study carried out b y TANAKA1, a ribonuclease was isolated from Streptomyces erythreus and exhibited a substrate specificity similar to ribonuclease T 1 which catalyzed the splitting of phosphodiester bonds of 3'-guanylic acid in RNA and the 2'-ester bond of guanosine 2',3'-cyclic phosphate. In addition, a further demonstration concerning the specificity of the former has been reported using yeast low molecular weight RNA as a substrate 2. An interest in the comparative biochemistry, particularly the protein structure, of ribonucleases which show the same substrate specificity prompted us to undertake the purification of a ribonuclease from S. erythreus, since the lack of a pure preparation of the ribonuclease has not permitted studies of its substantial properties and kinetics. The present report is concerned with studies on the purification and characterization of the physical and chemical properties of ribonuclease from S. erythreus. Some discussion will be concerned with a comparison of the amino acid composition of the ribonuclease with those of ribonuclease T 1 and bovine pancreatic ribonuclease (ribonuclease A).
Biochim. Biophys. Acta, 228 (1971) 636-647
PURIFICATION AND PROPERTIES OF RIBONUCLEASE S t
637
MATERIALS AND METHODS
DEAE-cellulose (o.82 mequiv./g) was purchased from Brown Co. and DEAES~phadex, QAE-Sephadex and Sephadex G-75 were obtained from Pharmacia, and yeast RNA used for ribonuclease assay was purchased from Kokoku Rayon and Pulp Co. (Tokyo).
Ribonuelease assay The enzymatic activity of ribonuclease was determined by measuring the absorbance at 260 m# of the acid-soluble digestion products from yeast RNA according to the method described by TANAKA3 using Tris-HC1 buffer instead of phosphate, and the specific activity (enzyme unit) was calculated in accordance with the definition presented previously by TANAKA3.
Electrophoretic analyses Polyacrylamide gel electrophoresis was performed in a Canalco analytical disc electrophoresis apparatus at room temperature with a 15 % cross-linked gel according to the procedure recommended by ORNSTEIN4 and DAVIS5. Gels were stacked at pH 7.5 with samples containing 200/~g of enzyme dissolved in 40 % sucrose tray buffer solution (pH 7.0) and were subjected to electrophoresis at 2.0 mA per gel. Gels were stained with 1 % amido black dissolved in 7 % acetic acid for I h and were electrophoretically de-stained (8 mA per gel) in 7 % acetic acid.
Ultracentri]ugal analysis A Hitachi model UCA-I ultracentrifuge was used to measure the sedimentation constant and to determine the molecular weight by sedimentation equilibrium. All analyses were performed in o.I M Tris-HC1 buffer, pH 7-4. Sedimentation velocity experiments were performed at 51 900 rev./min in a synthetic boundary cell. The sedimentation constant was corrected to a value corresponding to water at 20 °. The sedimentation equilibrium procedure was done with a double sector centerpiece at 20 350 rev./min. Schlieren images and Rayleigh interference fringes were recorded on Kodak metallographic plates, a n d the positions of the patterns were analyzed in a microcomparator (Shimadzu, Model SR-2). The partial specific volume of the protein was calculated from its amino acid composition by the method of COHN AND EDSALL e.
Amino acid analyses A sample of enzyme (approx. 2 mg) was suspended in I ml of glass-distilled HC1, de-aerated and sealed, and hydrolyzed at 11o% Immediately prior to analysis, hydrolyzates were evaporated to dryness under reduced pressure at 55 ° in a rotary evaporator. Amino acid analyses were performed in a Jeol's analyser model JLC-3BC (Japan Electron Optics Lab. Co., Tokyo) with optimal precision in the 0.05 #mole range. Separate samples were oxidized with performic acid for the determination of cysteic acid by the procedure of HIRS~. Tryptophan was estimated spectrophotometrically in an unhydrolyzed sample by the method of GOODWlN AND MORTONs and the method of SPIES and CHAMBERS~'1° using p-dimethylaminobenzaldehyde. Biochim. Biophys. Acta, 228 (1971) 636--647
~. YOSHIDAet al.
638
Molecular 7e,eight determination by gel/iltration The method of WHITAKER11 was applied to the purified ribonuelease. A column (1.6 cm × 88 cm) of Sephadex G-75 was equilibrated at room temperature with a buffer, pH 6.o, composed of o.I M sodium acetate and o.I M NaG1 (J = o.194 ). To measure the void volume (V0) of the column, blue dextran 2ooo was used. Other markers were bovine serum albumin (tool. wt. 7 ° ooo), pepsin (tool. wt. 35 ooo), ~-chymotrypsin (mol. wt. 25 ooo), bovine pancreatic ribonuelease (mol. wt. 14 ooo). 2-ml fractions were collected and monitored at 28o m/t, and the flow rate was regulated to 3o ml/h.
RESULTS
Purification procedure Step I and Step II (Table I) were performed according to TANAKA'S1 method and further steps (Steps I I I , IV and V) were followed by sequential chromatography at 4 ° . TABLE I SUMMARY OF PURIFICATION OF RIBONUCLEASE FROM S.
Step and treatment
Starting broth Acrinol p r e c i p i t a t e Batchwise DEAE-cellulose treatment III. DEAE-cellulose chromatography IV. D E A E - S e p h a d e x (A-25) chromatography V. Q A E - S e p h a d e x (A-5o ~ chromatography I. II.
Total protein
erythreus .Specific activity (units/A 280 mu)
Puri[ication factor
Yield (%)
IOO 1 13o g 2o.8 g
3° 270 12oo
9 4°
ioo 4° 29
55 ° m g 142 m g
125oo 45000
42o I5oo
io 6.9
lO6 m g
52000
i83o
6.o
Step I I I chromatography on DEAE-cellulose. The fraction treated batchwise with DEAE-cellulose was purified by DEAE-cellulose chromatography, the column being developed with o.i M acetate buffer, pH 4.0, containing 0.03 M NaC1 until an active fragment was eluted (Fig. I). The active effluents were pooled and the enzyme was subsequently concentrated by the addition of (NH4)2SO a to 66 °,o saturation. The precipitate produced during one day was dissolved in a minimal volume of distilled water, dialyzed against distilled water and lyophilized. Step I V chromatography on DEAE-Sephadex. The crude enzyme obtained in Step III, 0.35 g, was dissolved in 35 ml of o.i M Tris-HC1 buffer containing o.I M NaCI at pH 8.o and placed on a DEAE-Sephadex (A-25) column, 2 . 6 c m × 3 o cm, equilibrated with the same buffer. An exponential gradient elution was carried out from o.I M NaC1 in the buffer to the buffer containing 0.3 M NaC1 with a 1.2-! mixing chamber. Fractions of 15 ml were collected at a flow rate of 60 ml/h. Fig. 2 shows the result of the gradient elution portion of this column step. Tile last protein peak eluted with this gradient system contained the ribonuclease activity. Fractions conBiochim. Biophys. Acta, 228 (1971) 636-647
639
PURIFICATION AND PROPERTIES OF RIBONUCLEASE S t
? 6
• I.0 I
> tJ
::L E o oo o4
pool
0.3
-0.6
o
~M o o c~ X
o
-(1;
.-o-
IO00
o2
G0 o4
QI
o O4 *t
3ooo
2oo0 Effluent ml
Fig. I. Chromatography of a batchwise DEAE-cellulose-treated fraction on a column, 2.6 cm × 50 cm, of DEAE-cellulose. A sample of 4 g in 4 ° ml of o.i M acetate, pH 4.o, was applied to the columt~. Effluent fractions of 18 nil were collected at a rate of i5o ml]h.
l
pool
]
I0
--
"t
~
i o., . . . 5 0.0
Cl'co~n.
1 0.8
~
Q4
~-
t°g i 000 Effluent
i500"
~000
o
ml
l~ig. 2. Chromatography of partially purified enzyme on DEAE-Sephadex (A-25). Column: 2.6 cm × 3 ° cm. Load: 35 ° mg of DEAE-cellulose chromatographically purified enzyme. Elution: exponential gradient from o.i M NaC1 in o.I M Tris-HCl buffer (pH 8.o) to o.3o M NaC1 in the buffer ~ i t h a 12oo m] mixing chamber, at the flow rate of 60 ml/h, 15 ml per fraction.
t a i n i n g t h e e n z y m a t i c a c t i v i t y w e r e p o o l e d , a d j u c t e d t o p H 4.0 a n d c o n c e n t r a t e d b y t h e a d d i t i o n of ( N H 4 ) 2 S 0 4 as i n S t e p I I I . A f t e r d i a l y s i s a n d l y o p h i l i z a t i o n , a p p r o x . I i O m g of t h e p r o t e i n w e r e o b t a i n e d , w i t h a s p e c i f i c a c t i v i t y of 45 ooo u n i t s . Step V chromatography on QA E-Sephadex. 255 m g of t h e e n z y m e p r e p a r a t i o n o b t a i n e d f r o m t h e p r e c e d i n g c h r o m a t o g r a p h y w a s d i s s o l v e d i n 25 m l of o.~ M T r i s HC1 b u f f e r , p H 7.4, plus o . I o M NaC1 a n d w a s c h r o m a t o g r a p h e d o n a Q A E - S e p h a d e x (A-5o) c o l u m n , 2.6 c m × 52 c m , e q u i l i b r a t e d w i t h t h e b u f f e r c o n t a i n i n g o . I o M NaC1. A g r a d i e n t e l u t i o n s y s t e m f r o m o . I o t o 0.27 M NaC1 w i t h a I-1 m i x i n g c h a m b e r w a s c a r r i e d o u t b y t h e s a m e m e t h o d a s i n S t e p I V . F r a c t i o n s of 15 m l w e r e c o l l e c t e d a t a
Biochim. Biophys. Acta, 228 (1971) 636-647
64o
~
N. YOSHIDAet al.
2.0 t3 *t
~'1.5 0 CO
c~ 2.0 x
Lo
° "=:L
.~0.5
O4
a4 ~ 500
1000
1500
2000
Effluent ml
o ID <1
Fig. 3. Final purification of ribonuclease from S. erythreus on Q A E - S e p h a d e x (A-5o). Column: 2.6 cm × 52 cm. Load: 255 m g of D E A E - S e p h a d e x c h r o m a t o g r a p h i c a l l y purified enzyme. Elution: exponential gradient f r o m o. I iV[ NaC1 in o. I M Tris-HC1 buffer, p H 7.4, to o.27 M NaC1 in the buffer w i t h a i-1 mixing chamber, a t the frow r a t e of 18 ml/h, 15 ml per fraction.
flow rate of approx. 18 ml/h. As shown in Fig. 3, the enzyme active fraction yielded a symmetrical peak (about 200 mg of lyophilized protein). Upon re-chromatography of the purified enzyme thus obtained, on QAESephadex with a one-step elution with o.17 M NaC1 in o.I M Tris-HC1 buffer (Fig. 4), or with the same gradient elution as described above (Step V), the enzyme was eluted as a single symmetrical peak with the respect to the enzymatic activity as well as to the protein. The specific activity was increased about i8oo-fold by the total purification for the five steps, i.e. from 5 ° ooo to 55 ooo units. Table I summarizes the results obtained when IOO 1 of starting broth were processed b y the purification procedure just outlined. The over-all yield of enzymatic activity in the described experiment was about 6 °/0 and approx. IOO mg of the purified protein was prepared.
/ o.31o |/
6°r 5.51-
0 O2L__j
~
~
/
/'
F | z~
o.~
40O Effluent ml Fig. 4. Re-chromatography of final purified preparation, Step V, on QAE-Sephadex A-5o. Column: 1.6 c m × 5 4 cm. Load: 36.5 mg of QAE-Sephadex chromatographically purified enzyme. Elution: o.I M T r i s - H C l buffer containing o.i 7 M NaCI, p H 8.0, at the flow rate of 5 ml/h, ,-o ml per fraction.
Biochim. Biophys. Acta, 228 (1971) 636-647
PURIFICATION AND PROPERTIES OF RIBONUCLEASE S t
641
Criteria o/purity Disc electrophoretic patterns. The disc electrophoretic patterns shown in Fig 5 represent the protein bands in the sequential fractions during purification, and the final product shows a single band as shown in Fig. 5c.
Fig. 5. Polyacrylamide gel electrophoretic patterns of fraction obtained during the isolation of ribonuclease from S. ervthreus. 15 % small-pore gel, running at p H 7.0, 2.0 mA per gel for i h, 20o/tg of protein. A.The active fraction from DEAE-cellulose chromatography step. 13. The active fraction from DEAE-Sephadex chromatography step. C. The final product after QAE-Sephadex chromatography step.
Ultracentri/ugal pattern. Fig. 6 shows the ultracentrifugal Schlieren pattern of a solution of purified enzyme in o.I M Tris-HC1 buffer, pH 7.4, at a protein concentration of 5.2 mg/ml. The enzyme showed a single symmetrical peak with a sedimentation coefficient of 1.61 S. In a separate experiment carried out at a protein concentration of 3.3 mg/ml, the same s20,w value was obtained.
Fig. 6. Ultracentrifuge patterns for sedimentation velocity experiments of ribonuclease from S. erythreus in a synthetic boundary cell at 51 90o rev./min and 21.3 °. The protein concentration is 5.z mg/ml in o.i M Tris-HCl buffer, p H 7.4. The pictures were taken at i (A), 30 (B) and 60 (C) min after reaching the final speed.
Molecular weight Gel /iltration method. The purified enzyme was passed through a Sephadex G-75 column, and the elution pattern was compared with several markers which Biochim. Biophys. Acta, 228 (1971) 636-647
N. YOSHIDA et al.
642
Ve/Vo ! 2 . 0 ~ I. 6
I"I 4.0
"~hymolrypsi n
I
I I I IBovineIum Sre 4.2 4.4 4.6 Logorithrnof Molecul¢,rWeight
~
4.8
i
Fig. 7. Relationship b e t w e e n Ve/Vo values and molecular weights of proteins used in determining the molecular weight of ribonuclease from S. erythreus.
showed a linear relation between Ve/Vo (Ve; elution volume of each marker; Vo, void volume) and the logarithms of the molecular weights for the reference proteins (Fig. 7). On the basis of this relationship and the Ve/Vo value of 2.08 obtained for the enzyme, a molecular weight, of approx. 12 ooo was found for the ribonuclease of S. erythreus. This molecular weight value is supported by the experimental result that the enzyme is dialyzable through a collodion bag (Sartorius Membranfilter) under reduced pressure while it is not dialyzable on a cellulose tubing (Visking). TABLE II AMINO ACID COMPOSITION OF HYDROLYZATE OF RIBONUCLEASE FROM S. erythreus
Residue
Lys His Arg Asp
Thr Set Glu Pro Gly Ala Cys Val Ile Leu Tyr Phe 1NH~
Total
Residues per
N (% of
zoo g o/ protein
total N)
Calc. residues /or tool. wt. 3:2000
2.27 2-43 12.9o 12.oo 6.81 2.27 16.6o 6.05 6.67 3.82 1.37 4 .68 2.72 4.98 I 1.6o 6.15 o.16
3.05 4.57 28.4 8.83 5.80 2.25 I 1.04 5.37 10.o6 4.62 1.15 4 .o6 2.07 3.78 6.12 3.60 3.28
2.13 2-13 6.o7 12.4 8.1o 3.14 I5. 5 7.49 14- I 6.39 1.6o 5.74 2.88 5.28 8.54 5.03 7.5
lO2.51
*
IO8.O 5
* E x c l u d i n g N H a. Biochim. Biophys. Acta, 228 (1971) 636-647
lO6.52"
Nearest intregral number o~ residues per molecule 2 2 6 12 8 3 16 8 14 6 2 6 3 5 9 5 lO7
643
PURIFICATION AND PROPERTIES OF RIBONUCLEASE S t
Amino acid composition. The amino acid composition of the ribonuclease is shown in Table II, and the molecular weight was calculated to be I I 921. Sedimentation equilibrium method. The molecular weight of the ribonuclease was estimated independently from its sedimentation behavior. The protein was analyzed by the sedimentation equilibrium method at several concentrations (I.4I, 1.175 and 0.82 mg/ml). The equilibrium concentration of the protein at the meniscus was determined according to LA BAR'S 12procedure. Semi-logarithmic plots of fringe displacement against the square of the centrifugal radius afforded straight lines: a typical plot is presented in Fig. 8. This linear relationship as well as the symmetric
I
l
I
I
I
I 50.0
I 51.0
T.40
T.~
o
T.oo
"~.8O
r=
I 47.0
I 48.0
I 49.0 r 2 (Gin a )
Fig. 8. S e d i m e n t a t i o n e q u i l i b r i u m n l t r a c e n t r i f u g a t i o n of ribonucIease f r o m S. erythreus of t h e initial c o n c e n t r a t i o n 1.175 m g / m l a t 2o 35o r e v . ] m i n a t 29.2 °. Plot of l o g a r i t h m of t h e a b s o l u t e fringe d i s p l a c e m e n t , l o g / , as a f u n c t i o n of t h e s q u a r e of radial d i s t a n c e f r o m t h e c e n t e r of r o t a t i o n , $,2.
boundary shape in the sedimentation velocity patterns supports the homogeneity of the ribonuclease in molecular weight. Using a partial specific volume of o.711 ml/g, calculated from the amino acid composition, molecular weights under these conditions computed from the data obtained from three different concentrations were almost independent of the enzyme concentration, giving I I 9005100. The results of these three independent methods are in good agreement, indicating a molecular weight, of approx. I I 900 for the ribonuclease of S. erythreus.
Isoelectric point To estimate the isoelectric point of the ribonuclease, an electrophoresis on cellulose acetate film (OXOID) was carried out. The ionic strength of all buffer solutions was o.I and the pH value ranged from 3.0 to 6.013. After drying and staining with Ponceau 3R (0.4 % aqueous solution containing 3 % trichloroacetic acid), a single stained band was produced, and the distance migrated from the origin was measured on strips carrying all the bands. The electrophoretic mobility of the enzyme as a function of pH is shown in Fig. 9, which represents an isoelectric point near p H 4.5. Biochim. Biophys. Acta, 228 (1971) 6 3 6 - 6 4 7
~. YOSHIDA et al.
644
+L5 0
\
+1.0
cO
+0.5 °~
0 Q O C
-0.5
0
~5
-I .0 !
2.0
I
3.0
I
4.0
|
,5.0
I
6.0
pH
Fig. 9. E l e c t r o p h o r e t i c m o b i l i t y of ribonuclease f r o m S. erythreus. E x p e r i m e n t s were p e r f o r m e d u n d e r c o n d i t i o n s in w h i c h t h e electrophoresis w a s r u n for 45 m i n w i t h 16. 7 V / c m (2.2 m A / c m ) a t 4 °, in either g l y c i n e - H C l b u f f e r ( O ) or a c e t a t e (O) a t a n ionic s t r e n g t h of o.i.
Amino acid composition The mean values of four analyses for 24 and 48 h (duplicate analyses at each hydrolysis time) are represented in Table II. No significant increase of valine and isoleucine was observed even after 72 h of hydrolysis. The numbers for threonine and serine are the values obtained from duplicate analyses of the 8-h hydrolysate. Half-cystine was independently determined as cysteic acid and its value was 1. 9 residues per molecule as compared with 1.6 residues obtained from Table I I on the un-oxidized protein. No methionine nor t r y p t o p h a n could be detected in a n y analysis. The analytical results summarized in Table I I account for lO3 O/,oof the weight of the ribonuclease and the total number of amino acid residues is lO 7. The sugar content in the enzyme was examined by the method of DUBOlS et al. 1~, and found to be nearly zero.
DISCU SSION
The isolation of this enzyme is a necessary step in verifying a correlation between the enzymatic function, particularly binding of substrate to the active site, and the chemical structure as compared with those of ribonuclease T 1. In the present paper, ribonuclease from S. erythreus has been purified b y sequential chromatography with anion-exchange cellulose and Sephadex. The enzyme preparation appears to be homogeneous by the following criteria: re-chromatography on QAE-Sephadex, polyacrylamide disc gel electrophoresis, cellulose acetate film electrophoresis, sedimentation velocity and the linearity of the sedimentation equilibrium plot. For convenience, it appears appropriate to assign a trivial name to the purified ribonuclease, which we propose to call ribonuclease St. The amino acid composition and some physicochemical parameters of the purified enzyme have been determined. A molecular weight of i i 900 has been obtained Biochim. Biophys. Acta, 228 (1971) 636-647
PURIFICATION
AND PROPERTIES
OF RIBONUCLEASE
645
St
by three independent methods, Sephadex G-75 gel filtration, sedimentation equilibrium and amino acid composition. The amino acid composition of ribonuclease St differs notably from those oI ribonuclease T 1 and the bovine pancreatic ribonuclease (ribonuclease A) as shown in Table III. There are several interesting features in Table III. First of all, methionine TABLE
III
AMINO ACID COMPOSITION OF RIBON!dCLEASE St~ T 1 AND A
A m i n o acid
N u m b e r o[ residues per molecule Ribonudease St
Lys His Arg Asp Thr Ser Glu Pro Gly Ala Cy~ Val Met Ile Leu Tyr Phe Trp Total
Ribonuclease T I
Ribonuclease A
2 2 6 12 8 3 16 8 14 6 2 6 o 3 5 9 .5 o
i 3 i 15 6 15 9 4 12 7 4 8 o 2 3 9 4 I
io 4 4 15 io 16 II 4 3 12 8 9 4 3 2 6 3 o
107
105
124
and tryptophan are absent from ribonuclease St so far examined, whereas one tryptophan residue was found in ribonuclease T 1 and 4 methionine residues per ribonuclease A molecule. As to the function of the tryptophan residue of ribonuclease 3"1,it has been recently published that inactivation was caused by N-bromosuccinimide oxidative modification 15 and by the use of 2-hydroxy-5-nitrobenzyl bromide ~6. However, it appears that the tryptophan in ribonuclease T~ has no direct role in the catalytic process, but may be important in maintaining the proper conformation of the active site. From the findings that ribonuclease St has one more phenylalanine residue than ribonuclease 3"1 and that the tryptophan residue is found at the hydrophobic region ( T y r - G l u - T r p - P r o - I l e - L e u ) 17 of ribonuclease T1, a prediction m a y be made that ribonuclease St contains the phenylalanine residue at the position corresponding to the t r y p t o p h a n in the sequence of ribonuclease T r Secondly, it is iloteworthy that among these ribonucleases there are apparently some differences in the amount of several amino acids, such as half-cystine, serine, arginine and proline. In several experiments with native and oxidized ribonuclease St, the half-cystine content of the enzyme was found to be lower than those of ribonuclease T 1 and ribonuclease A. The number of half-cystine residue of ribonuclease St corresponds to a single disulfide bond per molecule, while in ribonuclease T 1 there Biochim. Biophys. Acta, 228 ( 1 9 7 1 ) 6 3 6 - 6 4 7
646
N. YOSHIDAet al.
are two disulfide bonds and there are 4 bonds in pancreatic ribonucleases isolated from bovine, porcine and rat sources TM. It is interesting that animal pancreatic ribonucleases show a more rigid secondary structure, owing to cross-linking b y the four disulfide linkages, compared with those of microbial ribonucleases, and that the two disulfide bridges in ribonuclease TI found in the fungus give a more rigid molecular construction than that of ribonuclease St in the bacterium, Streptomyces. Ribonuclease St exhibits a markedly different amino acid composition from the other ribonucleases; in spite of having the same per cent of threonine residues, the serine content of the enzyme is somewhat lower than that of the others and, in contrast, the arginine content is extremely high. The fact that ribonuclease St, an arginine-rich protein, is isoelectric near p H 4.5 suggests that at higher p H regions than the isolelectric point, the greater part of side chains of Asx and Glx residues in the protein exist not as amidation states nor as salt linkages formed between anionic groups, but as carboxylate ions. Finally, the amino acid composition of ribonuclease St shows a high proline content (7.5 residues per cent) which is twice that of ribonuclease T v The ~-helix coil of a protein m a y be disrupted at the points of the proline residues, and it hasbeen estimated that a protein with 8 °/o proline randomly distributed throughout the polypeptide chain would have an almost complete absence of ~-helix conformation '". Hence ribonuclease St would be expected to have either a low helical content or proline-rich regions of considerable size. On the other hand, it is suggested that ribonuclease T~ has a considerably regular conformation compared with ribonuclease St, since the content of proline residues of the former is approx. 4 %. The helical content of ribonuclease St, in fact, could not be estimated from our preliminary results of the optical rotatory dispersion and circular dichroism of the enzyme (N. YosrnDA, K. KURIYAMA,T. IWATA AND H. OTSUKA, unpublished) while the content of ribonuclease T 1 was calculated to be 20 to 30 % b y the Moffit-Yang method ~°. A more detailed comparison must await the outcome of amino acid sequence studies in progress with ribonuclease St.
ACKNOWLEDGMENTS
The authors wish to t h a n k Dr. K. T a n a k a for his kind advice of the purification procedure.
REFERENCES i K. TANAKA,J. Biochem. Tokyo, 5 ° (1961) 62. 2 K. TANAKA AND G. L. CANTONI,Bichim. Biophys. Acta, 72 (1963) 641. 3 K. TANAKA,in G. L. CANTONI AND D. R. DAVIS, Procedures in Nucleic Acid Research, H a r p e r a n d Row, N e w Y o r k , 1966, p. 14. 4 L. ORNSTEIN, Ann. N. Y. Acad. Sci., 121 (1964) 321. 5 B. J. DAVIS, Ann. N. Y. Acad. Sci., 121 (1964) 4o4 . 6 E. J. COliN AND J. T. EDSALL, Proteins, Amino Acids and Peptides, R e i n h o l d , N e w Y ork, 1943, p. 375. 7 C. H. W. HIRs, J. Biol. Chem., 219 (1956) 611. 8 T. W. G o o n w I N AND R. A. MORTON, Biochem. J., 4 ° (1946) 628. 9 J. R. SPIES AND D. C. CHAMBERS, Anal. Chem., 20 (1948) 3 o. i o J. R. SPIES AND D. C. CHAMBERS, Anal. Chem., 21 (1949) 1249.
Biochim. Biophys. Acta, 228 (1971) 636-647
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