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An agar-diffusion method for quantitative determination of ribonuclease
BIOCHIMICA ET BIOPHYSICA ACTA
463
BBA 97028
AN A G A R - D I F F U S I O N METHOD F O R Q U A N T I T A T I V E D E T E R M I N A T I O N OF R I B O N U C L E A S E C. H. T A N
Department o[ Biochemistry, Faculty o[ .Vledicine, University o[ Singapore, Singapore 3 (Republic o[ Singapore) (Received J u n e 3rd, 1971)
SUMMARY
i. A sensitive agar-diffusion slide method has been developed for the assay of ribonuclease activity in microvolumes of samples. 2. The method involves the diffusion of ribonuclease from small wells cut into an RNA-agar-coated microscopic glass slide. 3. Diffusion of ribonuclease results in the production of distinct zones of clearing aiound the wells after acid precipitation of the unhydrolysed RNA. 4. Conditions for optimal ribonuclease activity on the agar-diffusion slides were determined and the assay was applied to crude tissue extracts.
INTRODUCTION
Many procedures have been developed for the assay of ribonuclease activity 1-s. These usually involve the precipitation of undegraded RNA and the measurement of acid-soluble products released from the breakdown of RNA 1,9-1~. Most of the methods are lengthy and are only suitable for comparatively pure enzyme extracts. The requirement of rather pure substrates and the possibility of acid breakdown of RNA are two other disadvantages associated with these methods. A simple and sensitive method for quantitative estimation of ribonuclease activity using only microvolumes of samples will be described. This procedure involves the precipitation of unhydrolysed RNA trapped in agar. The hydrolysis of RNA b y ribonuclease produces zones of clearing on the agar which can be measured accurately. This procedure can be conveniently carried out as a thin-layer technique on microscopic glass slides. A similar method has been described recently for the assay of deoxyribonuclease ~4. MATERIALS AND METHODS
Preparation o/RNA-agar plates 3 ml of a hot solution of 1 % agar (Ionagar No. 2, Oxoid) in o.I M Tris-HC1 (pH 7.5) containing I mg/ml yeast RNA (Commercial grade, Type IIS, Sigma Chemical Co., reprecipitated twice with ethanol) were spread evenly on a 3 inch × I inch microscopic glass slide (previously cleaned and heated for 2 h at 18o°). The R N A agar was allowed to set at room temperature. A thin steel tube was used to bore 3-5 evenly spaced wells of 2.1 m m diameter on the agar. Biochim. Biophys. Acta, 247 (1971) 463-467
464
c . H . TAN
Measurement o] ribonudease activity To each well of the R N A - a g a r plate 4 #1 of a bovine pancreatic ribonuclease preparation (Type X I I - A , Sigma Chemical Co.) were added. The R N A - a g a r slides were placed in a plastic box containing moist blotting paper and incubated at known temperatures for periods up to 18 h. The slides were flooded with a mixture of I.O ~o u r a n y l acetate and IO % trichloroacetie acid (I : I, v/v) for I rain and then washed with water. T h e y were then placed against a dark background. Duplicate measurement of the diameter of the zones of clearing around the wells were made at right angles to one another (Fig. I) and the mean values obtained from them. To show the zones of hydrolysis more distinctly, the R N A t r a p p e d on the agar can be stained with o.oi % pyronin G (E. Merck, Darmstadt).
/ / / / / / / / / / / / / / / R N A - A G A R - C O A T E D GLASS SLIDE
/ / / / / / / / / / / / / / / Fig. I. Measurement of ribonuclease activity on an agar-diffusion slide. The diameter of the well is represented by y and the diameter of the zone of clearing represented by d. EXPERIMENTS
AND RESULTS
Conditions/or optimal ribonuclease activity on RNA-agar plates (r) Temperature. The effect of temperature on ribonuclease activity was investigated using two different concentrations of enzyme. Ribonuclease activity was measured b y the difference (D) between the diameter of the zone of clearing (d) and the diameter of the well (y). Optimal activity of ribonuelease was found to be at a temperature of 45 ° for the two ribonuclease concentrations used (Fig. 2). (2) Time course o/reaction. The rate of R N A hydrolysis, as measured b y the square of the diameter of the zone of clearing minus the square of the diameter of the well, was found to be linear during the first 4 h of incubation and decreased with time (Fig. 3)(3) Enzyme concentration. The effect of enzyme concentration on ribonuclease activity was investigated over three different periods of incubation (Fig. 4). W h e n the logarithms of different concentrations of ribonuclease were plotted against ribonuclease activity (D), a linear relationship was obtained. The coefficients of variation for a series of determinations of zone diameters at 0. 5/~g/ml and 5 °/~g/ml of standard ribonuclease were found to be approximately I . I °/o and 1. 4 °/o, respectively.
I3iochim. Biophys. Acta, 247 (197I) 463-467
465
DETERMINATION OF RIBONUCLEASE
25C
lO
20C
8
j
/
i1-.~11
O(mrn 15C
.j°z°-.\.\.
E E 100
°~o ?b °
4b ° 5,0° 6b ° Ternperoture
HI
"'~
7b °
50
/
/
o~
/
/
~
8 1~ Time (h)
1~
20
Fig. 2. E f f e c t of t e m p e r a t u r e on r i b o n u c l e a s e a c t i v i t y . I n c u b a t i o n c o n d i t i o n s are described in MATERIALS AND METHODS e x c e p t t h a t t h e period of i n c u b a t i o n w a s I h a n d t h e t w o r i b o n u c l e a s e c o n c e n t r a t i o n s were 0. 5/~g/ml ( O - O ) a n d 5o/~g/ml ( 0 - 0 ) . E a c h circle r e p r e s e n t s a m e a n of three determinations. Fig. 3. T i m e course of r e a c t i o n a t 45 ° in t h e p r e s e n c e of 0. 5/~g]ml of ribonuclease. O t h e r incub a t i o n c o n d i t i o n s are g i v e n in MATERIALS AND METHODS. E a c h p o i n t on t h e c u r v e r e p r e s e n t s a m e a n of t h r e e d e t e r m i n a t i o n s .
14 12
~,.
O(mm)
log ribonuclease concn. (ng/ml)
Fig. 4. E f f e c t of e n z y m e c o n c e n t r a t i o n on r i b o n u c l e a s e a c t i v i t y a t 45 ° for i h ( O - @ ) , 2 h (•-• ) a n d 4 h ( & - &). O t h e r c o n d i t i o n s of i n c u b a t i o n are described in MATERIALS AND METHODS E a c h p o i n t on t h e g r a p h r e p r e s e n t s a m e a n of t h r e e d e t e r m i n a t i o n s .
Application o/ the agar-di//usion technique to the assay o/ribonuclease activity in crude tissue extracts and sera Crude rat tissue extracts (IO % w e t w t . / v o l . ) i n o.oi M Tris-HC1 (pH 7.5) were prepared and assayed for ribonuclease activity by the agar-diffusion technique. T h e r e s u l t s ( T a b l e I) s h o w e d t h a t t h e t e s t i s e x t r a c t c o n t a i n e d t h e l o w e s t r i b o n u c l e a s e a c t i v i t y w h i l e t h e p a n c r e a s e x t r a c t h a d a IOOO-fold h i g h e r a c t i v i t y t h a n t h a t o f t h e testis. In a similar manner
ribonuclease
activity
was assayed
in sera obtained
from
Biochim. Biophys. Acta, 247 (1971) 463-467
466
c.H. TAN
]'ABLE
I
R 1 B O N U C L E A S E A C T I V I T Y IN C R U D E RAT T I S S U E E X T R A C T S
A s s a y s of r i b o n u c l e a s e a c t i v i t y w e r e c a r r i e d o u t a t 45 ° f o r i h. O t h e r c o n d i t i o n s a r e d e s c r i b e d in MATERIALS AND METHODS
"l'issue
Ribonuclease activity D (ram) *
Speci/ic activity o/ribonuclease (ttg/mg protein)*
Liver Spleen Pancreas Heart
3-95 4.45 7.20 3-35
0.02 0.04 I o. 7 ° o.o2
Testis
3.20
O.Ol
* R e s u l t s a r e a n a v e r a g e of d u p l i c a t e s a m p l e s .
2o normal h u m a n subjects. The mean value obtained for D was 5.o5 m m ( ± o . I 5 S.E.) which corresponded to a value of a p p r o x i m a t e l y 49 ° ng of ribonuclease or 4.9" IO 2 Kunitz unit/ml of serum. DISCUSSION
The present agar-diffusion method described for the measurement of ribonuclease activity tlas several advantages, namely, simplicity, sensitivity, rapidity and good reproducibility. Assays can be carried out rapidly and conveniently on agarcoated glass slides. No elaborate instrumentation is required here except for the use of an accurate device for the measurement of the diameter of the circular zones of clearing as a result of R N A hydrolysis, lV[easurements can be made immediately after the precipitation of unhydrolysed RNA. Precipitating agents are less i m p o r t a n t here since I M HC1 can replace the uranyl acetate-trichloroacetate mixture with equally satisfactory results. Moreover, there is no necessity for the use of highly purified R N A as substrate. The present method also dispenses with the use of tissue blanks and substrate blanks which are essential to m a n y other methods since the tissues as well as the substrate (if not highly purified) contribute a significant a m o u n t of acid-soluble products 1. This method allows samples of as little as 4 #1 to be assayed for ribonuclease activity and is particularly useful when small amounts of tissue extracts are available for analysis; this being very pertinent to small-scale cell cultures. Here the total n u m b e r of cells m a y be tile limiting factor so t h a t it m a y be necessary to decrease the volume of the extracts in older to increase the enzyme concentration. The procedure is sensitive and can detect ribonuclease in the region of 4" 1o-5/~g and, in this respect, compares favourably with tlie method of DICKMAN el al. 12 or t h a t of AMBELLAN AND HOLLANDER11. Since this method can be used to detect ribonuclease activity on crude tissue extracts, it would prove to be particularly helpful in rapidly monitoring the enzyme during its purification. ACKNOWLEDGEMENTS
The author wishes to t h a n k Misses F. H. Ng and B. K. Gwee for their skillful technical assistance and Miss J. Chung for t y p i n g the manuscript. The author is grateful to Dr. S. E. Aw for critical reading of the manuscript. Biochim. Biophys. Acla, 2 4 7 (197 I) 4 6 3 - 4 6 7
DETERMINATION OF RIBONUCLEASE
467
REFERENCES I J. S. ROTH, in H. BuscH, Methods in Cancer Research, Vol. 3, Academic Press, New York. 1967, p. 153. 2 M. KUNITZ, J. Biol. Chem., 164 (1946) 563. 3 J. A. BAIN AND I-I. P. RUSCH, J. Biol. Chem., 153 (1944) 6594 ~V[. McCARTY, J. Exp. Med., 88 (1948) 181. 5 E. iV[. CROOK, A. P. MATHIAS AND B. R. RABIN, Biochem. J., 74 (196o) 234 6 R. SHAPIRO, Anal. Bioehem., 3 (1962) 308. 7 S. B. ZIMMERMAN AND G-. SANDEEN, Anal. Biochem., io (1965) 444. 8 F. ]V[OLEMANS, M. VAN MONTAGU AND W. FIERS, Eur. J. Biochem., 4 (1968) 524 . 9 C. B. ANFINSEN AND F. ]-[. VV~HITE, JR., in P. D. BOYER, H. LARDY AND t{. MYRBACK, The Enzymes, Vol. 5, Academic Press, N e w York, 1961, p. 95. IO L. JOSEFSSON AND S. LAGERSTEDT, in D. GLICK, Methods o/ Biochemical Analysis, Vol. 9, Wiley (Interscience), N e w York, 1962, p. 39I i E. AMBELLAN AND V. P. HOLLANDER, Anal. Biochem., 17 (1966) 474. 12 S. R. DICKMAN, J. P. AROSKAR AND R. g . KROPF, Biochim. Biophys. dcta, 21 (I956) 539. 13 W. C. SCHNEIDER AND G. H. HOGEBOOM, J. Biol. Chem., 198 (1952) 155. 14 A. W. JARVIS AND R. C. LAWRENCE, Can. J. Biochem., 47 (1969) 673.
Biochim. Biophys. Acta, 247 (i97 I) 463 467
463
BBA 97028
AN A G A R - D I F F U S I O N METHOD F O R Q U A N T I T A T I V E D E T E R M I N A T I O N OF R I B O N U C L E A S E C. H. T A N
Department o[ Biochemistry, Faculty o[ .Vledicine, University o[ Singapore, Singapore 3 (Republic o[ Singapore) (Received J u n e 3rd, 1971)
SUMMARY
i. A sensitive agar-diffusion slide method has been developed for the assay of ribonuclease activity in microvolumes of samples. 2. The method involves the diffusion of ribonuclease from small wells cut into an RNA-agar-coated microscopic glass slide. 3. Diffusion of ribonuclease results in the production of distinct zones of clearing aiound the wells after acid precipitation of the unhydrolysed RNA. 4. Conditions for optimal ribonuclease activity on the agar-diffusion slides were determined and the assay was applied to crude tissue extracts.
INTRODUCTION
Many procedures have been developed for the assay of ribonuclease activity 1-s. These usually involve the precipitation of undegraded RNA and the measurement of acid-soluble products released from the breakdown of RNA 1,9-1~. Most of the methods are lengthy and are only suitable for comparatively pure enzyme extracts. The requirement of rather pure substrates and the possibility of acid breakdown of RNA are two other disadvantages associated with these methods. A simple and sensitive method for quantitative estimation of ribonuclease activity using only microvolumes of samples will be described. This procedure involves the precipitation of unhydrolysed RNA trapped in agar. The hydrolysis of RNA b y ribonuclease produces zones of clearing on the agar which can be measured accurately. This procedure can be conveniently carried out as a thin-layer technique on microscopic glass slides. A similar method has been described recently for the assay of deoxyribonuclease ~4. MATERIALS AND METHODS
Preparation o/RNA-agar plates 3 ml of a hot solution of 1 % agar (Ionagar No. 2, Oxoid) in o.I M Tris-HC1 (pH 7.5) containing I mg/ml yeast RNA (Commercial grade, Type IIS, Sigma Chemical Co., reprecipitated twice with ethanol) were spread evenly on a 3 inch × I inch microscopic glass slide (previously cleaned and heated for 2 h at 18o°). The R N A agar was allowed to set at room temperature. A thin steel tube was used to bore 3-5 evenly spaced wells of 2.1 m m diameter on the agar. Biochim. Biophys. Acta, 247 (1971) 463-467
464
c . H . TAN
Measurement o] ribonudease activity To each well of the R N A - a g a r plate 4 #1 of a bovine pancreatic ribonuclease preparation (Type X I I - A , Sigma Chemical Co.) were added. The R N A - a g a r slides were placed in a plastic box containing moist blotting paper and incubated at known temperatures for periods up to 18 h. The slides were flooded with a mixture of I.O ~o u r a n y l acetate and IO % trichloroacetie acid (I : I, v/v) for I rain and then washed with water. T h e y were then placed against a dark background. Duplicate measurement of the diameter of the zones of clearing around the wells were made at right angles to one another (Fig. I) and the mean values obtained from them. To show the zones of hydrolysis more distinctly, the R N A t r a p p e d on the agar can be stained with o.oi % pyronin G (E. Merck, Darmstadt).
/ / / / / / / / / / / / / / / R N A - A G A R - C O A T E D GLASS SLIDE
/ / / / / / / / / / / / / / / Fig. I. Measurement of ribonuclease activity on an agar-diffusion slide. The diameter of the well is represented by y and the diameter of the zone of clearing represented by d. EXPERIMENTS
AND RESULTS
Conditions/or optimal ribonuclease activity on RNA-agar plates (r) Temperature. The effect of temperature on ribonuclease activity was investigated using two different concentrations of enzyme. Ribonuclease activity was measured b y the difference (D) between the diameter of the zone of clearing (d) and the diameter of the well (y). Optimal activity of ribonuelease was found to be at a temperature of 45 ° for the two ribonuclease concentrations used (Fig. 2). (2) Time course o/reaction. The rate of R N A hydrolysis, as measured b y the square of the diameter of the zone of clearing minus the square of the diameter of the well, was found to be linear during the first 4 h of incubation and decreased with time (Fig. 3)(3) Enzyme concentration. The effect of enzyme concentration on ribonuclease activity was investigated over three different periods of incubation (Fig. 4). W h e n the logarithms of different concentrations of ribonuclease were plotted against ribonuclease activity (D), a linear relationship was obtained. The coefficients of variation for a series of determinations of zone diameters at 0. 5/~g/ml and 5 °/~g/ml of standard ribonuclease were found to be approximately I . I °/o and 1. 4 °/o, respectively.
I3iochim. Biophys. Acta, 247 (197I) 463-467
465
DETERMINATION OF RIBONUCLEASE
25C
lO
20C
8
j
/
i1-.~11
O(mrn 15C
.j°z°-.\.\.
E E 100
°~o ?b °
4b ° 5,0° 6b ° Ternperoture
HI
"'~
7b °
50
/
/
o~
/
/
~
8 1~ Time (h)
1~
20
Fig. 2. E f f e c t of t e m p e r a t u r e on r i b o n u c l e a s e a c t i v i t y . I n c u b a t i o n c o n d i t i o n s are described in MATERIALS AND METHODS e x c e p t t h a t t h e period of i n c u b a t i o n w a s I h a n d t h e t w o r i b o n u c l e a s e c o n c e n t r a t i o n s were 0. 5/~g/ml ( O - O ) a n d 5o/~g/ml ( 0 - 0 ) . E a c h circle r e p r e s e n t s a m e a n of three determinations. Fig. 3. T i m e course of r e a c t i o n a t 45 ° in t h e p r e s e n c e of 0. 5/~g]ml of ribonuclease. O t h e r incub a t i o n c o n d i t i o n s are g i v e n in MATERIALS AND METHODS. E a c h p o i n t on t h e c u r v e r e p r e s e n t s a m e a n of t h r e e d e t e r m i n a t i o n s .
14 12
~,.
O(mm)
log ribonuclease concn. (ng/ml)
Fig. 4. E f f e c t of e n z y m e c o n c e n t r a t i o n on r i b o n u c l e a s e a c t i v i t y a t 45 ° for i h ( O - @ ) , 2 h (•-• ) a n d 4 h ( & - &). O t h e r c o n d i t i o n s of i n c u b a t i o n are described in MATERIALS AND METHODS E a c h p o i n t on t h e g r a p h r e p r e s e n t s a m e a n of t h r e e d e t e r m i n a t i o n s .
Application o/ the agar-di//usion technique to the assay o/ribonuclease activity in crude tissue extracts and sera Crude rat tissue extracts (IO % w e t w t . / v o l . ) i n o.oi M Tris-HC1 (pH 7.5) were prepared and assayed for ribonuclease activity by the agar-diffusion technique. T h e r e s u l t s ( T a b l e I) s h o w e d t h a t t h e t e s t i s e x t r a c t c o n t a i n e d t h e l o w e s t r i b o n u c l e a s e a c t i v i t y w h i l e t h e p a n c r e a s e x t r a c t h a d a IOOO-fold h i g h e r a c t i v i t y t h a n t h a t o f t h e testis. In a similar manner
ribonuclease
activity
was assayed
in sera obtained
from
Biochim. Biophys. Acta, 247 (1971) 463-467
466
c.H. TAN
]'ABLE
I
R 1 B O N U C L E A S E A C T I V I T Y IN C R U D E RAT T I S S U E E X T R A C T S
A s s a y s of r i b o n u c l e a s e a c t i v i t y w e r e c a r r i e d o u t a t 45 ° f o r i h. O t h e r c o n d i t i o n s a r e d e s c r i b e d in MATERIALS AND METHODS
"l'issue
Ribonuclease activity D (ram) *
Speci/ic activity o/ribonuclease (ttg/mg protein)*
Liver Spleen Pancreas Heart
3-95 4.45 7.20 3-35
0.02 0.04 I o. 7 ° o.o2
Testis
3.20
O.Ol
* R e s u l t s a r e a n a v e r a g e of d u p l i c a t e s a m p l e s .
2o normal h u m a n subjects. The mean value obtained for D was 5.o5 m m ( ± o . I 5 S.E.) which corresponded to a value of a p p r o x i m a t e l y 49 ° ng of ribonuclease or 4.9" IO 2 Kunitz unit/ml of serum. DISCUSSION
The present agar-diffusion method described for the measurement of ribonuclease activity tlas several advantages, namely, simplicity, sensitivity, rapidity and good reproducibility. Assays can be carried out rapidly and conveniently on agarcoated glass slides. No elaborate instrumentation is required here except for the use of an accurate device for the measurement of the diameter of the circular zones of clearing as a result of R N A hydrolysis, lV[easurements can be made immediately after the precipitation of unhydrolysed RNA. Precipitating agents are less i m p o r t a n t here since I M HC1 can replace the uranyl acetate-trichloroacetate mixture with equally satisfactory results. Moreover, there is no necessity for the use of highly purified R N A as substrate. The present method also dispenses with the use of tissue blanks and substrate blanks which are essential to m a n y other methods since the tissues as well as the substrate (if not highly purified) contribute a significant a m o u n t of acid-soluble products 1. This method allows samples of as little as 4 #1 to be assayed for ribonuclease activity and is particularly useful when small amounts of tissue extracts are available for analysis; this being very pertinent to small-scale cell cultures. Here the total n u m b e r of cells m a y be tile limiting factor so t h a t it m a y be necessary to decrease the volume of the extracts in older to increase the enzyme concentration. The procedure is sensitive and can detect ribonuclease in the region of 4" 1o-5/~g and, in this respect, compares favourably with tlie method of DICKMAN el al. 12 or t h a t of AMBELLAN AND HOLLANDER11. Since this method can be used to detect ribonuclease activity on crude tissue extracts, it would prove to be particularly helpful in rapidly monitoring the enzyme during its purification. ACKNOWLEDGEMENTS
The author wishes to t h a n k Misses F. H. Ng and B. K. Gwee for their skillful technical assistance and Miss J. Chung for t y p i n g the manuscript. The author is grateful to Dr. S. E. Aw for critical reading of the manuscript. Biochim. Biophys. Acla, 2 4 7 (197 I) 4 6 3 - 4 6 7
DETERMINATION OF RIBONUCLEASE
467
REFERENCES I J. S. ROTH, in H. BuscH, Methods in Cancer Research, Vol. 3, Academic Press, New York. 1967, p. 153. 2 M. KUNITZ, J. Biol. Chem., 164 (1946) 563. 3 J. A. BAIN AND I-I. P. RUSCH, J. Biol. Chem., 153 (1944) 6594 ~V[. McCARTY, J. Exp. Med., 88 (1948) 181. 5 E. iV[. CROOK, A. P. MATHIAS AND B. R. RABIN, Biochem. J., 74 (196o) 234 6 R. SHAPIRO, Anal. Bioehem., 3 (1962) 308. 7 S. B. ZIMMERMAN AND G-. SANDEEN, Anal. Biochem., io (1965) 444. 8 F. ]V[OLEMANS, M. VAN MONTAGU AND W. FIERS, Eur. J. Biochem., 4 (1968) 524 . 9 C. B. ANFINSEN AND F. ]-[. VV~HITE, JR., in P. D. BOYER, H. LARDY AND t{. MYRBACK, The Enzymes, Vol. 5, Academic Press, N e w York, 1961, p. 95. IO L. JOSEFSSON AND S. LAGERSTEDT, in D. GLICK, Methods o/ Biochemical Analysis, Vol. 9, Wiley (Interscience), N e w York, 1962, p. 39I i E. AMBELLAN AND V. P. HOLLANDER, Anal. Biochem., 17 (1966) 474. 12 S. R. DICKMAN, J. P. AROSKAR AND R. g . KROPF, Biochim. Biophys. dcta, 21 (I956) 539. 13 W. C. SCHNEIDER AND G. H. HOGEBOOM, J. Biol. Chem., 198 (1952) 155. 14 A. W. JARVIS AND R. C. LAWRENCE, Can. J. Biochem., 47 (1969) 673.
Biochim. Biophys. Acta, 247 (i97 I) 463 467