ANALYTICAL
BIOCHEMISTRY
A Method
loI&
233-239
(1979)
for the Determination Activity
of Alkaline Ribonuclease in Human Serum
(EC 3.1.4.22)
P. H. SCOTT Biochemistry
Department, Selly Oak Hospital, Birmingham
29, United Kingdom
Received January 11, 1979 The development and evaluation of a method for the determination of alkaline ribonuclease (RNase; EC 3.1.4.22) activity in human serum is described. Transfer RNA at a concentration of 4.0 g/liter is used as substrate. The effects of monovalent and divalent cations, phosphate, and EDTA have been studied. The method is standardized with cytidine, and appropriate units of activity are defined. It is linear up to an activity of 800 units/liter. The within batch coefficient of variation was 4.%, and between batch it was 6.0%. Age-related normal values of serum alkaline RNase activity in infants, children, and adults are given.
Since the first demonstration of RNase activity in blood by Zittle and Reading (I), there have been numerous studies involving the determination of the activity of the serum enzyme in normal and pathological conditions. The increase in RNase with uremia is well known (2,3); however, its association with carcinoma is less well established. Both normal (45) and elevated enzyme activities (6,7) have been claimed. Whether these disparate observations result from histological dissimilarities in the tumors as suggested by Chretien et al. (8) or from methodological inadequacies remains unresolved. Recently it has been confirmed that serum alkaline RNase activity can be related to nitrogen balance (9) and thereby becomes a valuable indicator of nutritional status, being considerably increased in malnutrition (10,ll). The potential of human serum alkaline RNase assays, particularly with respect to more delicate changes in activity, remains to be explored. It has been shown that in normal low-birth-weight babies the serum enzyme correlates well with nitrogen retention (12), and that differences in enzyme activity can be related to the nature of the dietary protein during early postnatal 233
life ( 13). Similarly, during pregnancy racial differences in human serum alkaline RNase levels have been found which were probably nutritional in origin (14) and which may allow identification of pregnancies which result in light-for-gestational-age babies (15). For studies such as these, a sensitive and optimized assay procedure is vitally important. Though turbidimetric (5,16) and colorimetric (17) methods have been developed, serum alkaline RNase activity is usually determined spectrophotometrically by measuring the absorbance of the short-chain oligonucleotides produced, after differential precipitation of substrate and products. Most use has been made of the methods devised by Ambellan and Hollander (18) and by Roth (19). These were, however, developed on and for use with tissue extracts and it is unlikely that they provide a valid optimized assay of human serum enzyme activity. Even among allegedly serum assays there exist many anomalies. The concentration of the RNA substrate used varies from 0.83 (20) to 13.3 g/liter (21); the optimum pH for the human serum enzyme ranges from 6.5 (3) to 8.5 (8); a variety of different ions have been used for activation of the enzyme. 0003-2697/79/180233-07$02.00/O Copyright 8 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
234
P. H. SCOTT
The mode of calibration and derivation of units of activity used in many of the methods is unacceptable. The simplest form relates enzyme activity to, and expresses it in terms of, the absorbance difference produced in the assay (820). Such an arbitrary system negates any form of true calibration, being dependent more on the optical system of the spectrophotometer and the sensitivity of the photodetector. The stability and comparative ease of purification of RNase has facilitated its use as a standard material for calibration of all types of assays (2,5,9,10,21). In most cases bovine pancreatic RNase has been used, but differences between bovine and human enzyme (22) render this material unsuitable. It is also unacceptable to express in mass terms (pg RNase/liter) that which is measured as an activity. Some method of calibration was therefore required which was more consistent with modern trends in enzyme analysis (23). As a consequence of the inadequacies of the methods currently available, it was opportune to reexamine particularly those characteristics of human serum alkaline RNase which are relevant to its assay, and from that to devise a method specifically suited to the human serum enzyme. METHOD
acetate in 70 ml distilled water; this was adjusted to pH 5.5 and made up to 100 ml with water. For use 12 ml of solution A was combined with 1 ml of solution B and with 9 ml distilled water. Cytidine calibration solution, 30 mmoY liter was prepared by dissolving 0.0729 g cytidine in distilled water to a volume of 10 ml. Procedure Into 3-ml polypropylene test tubes was pipetted 300 ~1 of buffered substrate, and into an equivalent number of similar test tubes was pipetted 300 ~1 of Tris-HCl buffer. For each batch of determinations enough tubes were set up for the tests, a calibration solution, a quality-control serum, and a substrate blank. To each tube was added 30 ~1 of 2.0 mol/liter sodium chloride solution. The tubes were then placed in a 37°C constant-temperature water bath for 5 min, and to each pair of tubes was added 10 ~1 of serum, test or control, or calibrant. After incubation for 60 min the reaction was terminated by addition of 2.5 ml of precipitating reagent. The solutions were chilled at -20°C for 10 min and were centrifuged at 3000g for 10 min. The supematants were measured spectrophotometrically against water at 260 nm in a lo-mm path-length silica cell.
Reagents
Calculation
Buffered substrate was prepared by dissolving 113 mg transfer RNA from bakers’ yeast (Boehringer Corp. (London) Ltd.) in 20 ml 0.04 mol/liter Tris-HCl buffer, pH 7.4, and then the solution was made up to 25 ml with buffer. Sodium chloride, 2.0 moYliter in distilled water. Precipitation reagent was prepared as two stock solutions. Solution A was prepared by dissolving 0.07 g lanthanum nitrate in 5 ml distilled water, the solution than being made up to 100 ml with anhydrous ethanol. Solution B was prepared by dissolving 5.58 g magnesium acetate and 8.20 g sodium
AOD26o = test ODzsO - (test blank OD260 + substrate blank OD,,,). Alkaline RNase activity (units/liter) =
of Results
AODm calibrant OD260 X
EVALUATION
[calibrant
(mmoYliter)] 0.06
OF ASSAY CONDITIONS
Choice of Substrate and Substrate Concentration Though there were advantages to be gained from the use of synthetic substrates,
DETERMINATION
OF ALKALINE
in general their inaccessibility precluded them. Polycytidylic acid has been employed (3,24) but, although it gives a highly sensitive assay, not all the isoenzymes of human serum alkaline RNase are equallly effective against the secondary phosphate esters of cytidine 3’-phosphate (25). Also, in the United Kingdom, the high cost of this substrate would prohibit its wide routine use. It was therefore decided to use naturally occurring yeast RNA, but to select the transfer RNA fraction which gave better assay precision (26). Two yeast transfer RNA products (tRNA(a) from Boehringer Corp. (London) Ltd., and tRNA(b) from Sigma Chemical Co., Ltd.) and one transfer RNA from Escherichia coli (tRNA(c) from B.D.H. Chemicals Ltd.) were examined. Both gave low blank values though only tRNA(a) conformed to Roth’s rigorous criteria of acceptability (19). The relationship of enzyme activity to substrate concentration was studied for each of the tRNAs over the range O-4.0 g/liter. The tRNA(b) substrate failed to give anything approaching a maximum velocity for the reaction. The Michaelis constants given by substrates tRNA(a) and tRNA(c), respectively, were 0.92 and 0.43 g/liter, demonstrating the low substrate affinity characteristic of enzymes which act on high molecular weight substrates. All substrates after reaction and precipitation gave acid-soluble oligonucleotide products of reaction with absorption maxima at 260 nm. The tRNA(a) substrate was chosen for use in the assay, at a concentration of 4.0 g/liter.
RNase IN HUMAN
235
SERUM
suggested that both acid (pH 6.5) and alkaline (pH 7.6) RNases exist. This was investigated over the pH range 6.2 to 7.8 using (i) 0.04 and 0.08 mol/liter imidazole (for pH 6.2 to 7.0) and Tris (for pH 7.05 to 7.8) buffers, and (ii) 0.055 mol/liter phosphate-borate buffer (equivalent in this assay to that used by Reddi (3,22)). The results are shown in Fig. 1. With the imidazole/Tris buffer combination two definite peaks of RNase activity, with optima at pH 7.06 and 7.4, were observed. The latter was little affected by increase in buffer concentration provided that sodium chloride was included in the assay solution; if this was absent then enzyme activity continued to increase with increasing buffer concentration. With the more acidic activity there was an increase associated with the higher buffer concentration. The phosphate-borate buffer, on the other hand, gave a single broad peak of activity which was a maximum at pH 6.86. As has been demonstrated (22) phosphate
500 ALKALINE RNASE ACTIVIN 400
UNITS/L
Effect of pH and Buffer Concentration There is some disagreement over the optimum of pH of human serum RNase activity. In tissue extracts both acid and alkaline RNases have been reported. In human serum Reddi (22) has indicated that only an acid RNase optimum at pH 6.5 is present, while Coombes et al. (27) have
7.0
pH
a"
FIG. 1. Effect of pH on the activity of human serum alkaline RNase using 0.04 moYliter imidazole/Tris (0 l ), 0.08 mol/liter imidazole/Tris (0 - - - 0), and 0.055 moYliter phosphate-borate (A A) buffers.
236
P. H. SCOTT
activates serum RNase activity. This has, however, been shown to be accompanied in the case of bovine pancreatic RNase with a shift in the pH optimum to the acid (28). The results obtained here indicate that this also happens with the human serum acid RNase, but that the alkaline RNase retains its higher pH though this becomes obscured by the acid RNase which undergoes greater phosphate activation. Because of this it was not thought appropriate to use phosphate buffer, or to include phosphate ion, in the assay procedure for human serum alkaline RNase. Tris-HCl buffer, pH 7.4 and at a concentration of 0.04 mol/liter was chosen. Effect of Monovalent and EDTA
and Divalent
Cations
A variety of cations, and EDTA, have been included in the reaction solutions for the assay of alkaline RNase activity but there has not yet been a comparative study of their effects. The monovalent cations lithium, sodium potassium, and ammonium (all with chloride as anion) were examined at concentrations from 0 to 350 mmollliter, and the divalent cations magnesium, calium, and cobalt (again with chloride as anion) were studied at concentrations from 0 to 53
500 400 ALKALINE 300 RNA% 200 ACTIVIN
. 100
UN!TS/L ILml
200
300
20
40
60
CATION CONCEhTRATIONW+YIOL/L
FIG. 2. Effects of the monovalent cations Na+ (X-X), K+ (mn ), Li+ (Ol ), and NH,+ (A - - - A), and the divalent cations Cazf (0 0), Mgz+ (U Cl), and Co*+ (A A) on human serum alkaline RNase activity.
mmol/liter. The results are shown in Fig. 2. All the cations showed an optimal concentration for maximum enzyme activity. The greatest activation was by sodium and potassium, and the former was chosen for inclusion in the assay at a concentration of 175 mmol/liter. Both Farkas (29) and Houck and Berman (5) had also added sodium but they used it at the lower concentration of 130 mmol/liter. There was no advantage in including a divalent cation as Biswas and Hindocha (20) have done. Addition of magnesium in the presence of 175 mmol/ liter sodium produced an immediate and progressive decrease in enzyme activity, probably due to competitive inhibition. The effect of EDTA was studied over the concentration range O-O.9 mmol/liter, using Tris salt to dissolve the EDTA so as to avoid addition of sodium or potassium. It was shown that EDTA has no effect on human serum alkaline RNase activity and its inclusion in the assay, as proposed by Shenkin et al. (26), was unnecessary. Calibration
of the Assay
Two factors determine the nature of the reaction products which are measured as an absorbance at 260 nm. The first is the specificity of the enzyme itself and the second is the selectivity of the precipitating reagent. Human serum alkaline RNase acts predominantly on the cytidine 3’-phosphate linkages, though certain of the isoenzymes show activity toward uridine 3’-phosphate linkages (25). Alkaline RNase activities of a series of serum specimens were redetermined and compared with the activities measured using polynucleotides as substrates in place of tRNA. With polyadenylic and polyguanylic acids there was no correlation, but a highly significant correlation was obtained with both polycytidylic and polyuridylic acids (Fig. 3). The activity shown with polycytidylic acid was much greater than that shown toward polyuridylic acid. Of the oligonucleotide fragments
DETERMINATION
OF ALKALINE
RNase IN HUMAN
cytidine per liter, stated conditions.
237
SERUM
per minute,
under the
Linearity
Using both a serum specimen with a high alkaline RNase activity and a purified preparation of the human serum enzyme, it was shown that the method was linear up to 800 units/liter. Sera with activities greater than this were repeated using a 5~1 sample. .
ACTIVITY
0.3
Precision
.
-
AOD'EONM
0.6
0.7 ALKALINE
0.8
0.9
1.0
RNASE ACTIVITY AODXONM
FIG. 3. The correlation of human serum alkaline RNase activity with the activity measured using polycytidylic acid (&) (r = 0.877, P < 0.001) and using polyuridylic acid (A) (r = 0.919, P < 0.001). Enzyme activity is expressed as AOD,,,.
resulting from hydrolysis of tRNA, the majority are therefore likely to be short sequences with cytidine as the terminal base. After hydrolysis, the oligonucleotide fragments are selectively precipitated so that only the acid-soluble products of up to five or six nucleotides in length remain in solution (30). The solubility of the uridylic acidcontaining fragments is less than that of the cytidylic acid-containing ones, so that precipitation favors the former. Cytidine 3’-phosphate was examined as a material for calibration of the assay but was found to be unsuitable. Cytidine however was shown to be a good substitute, being highly soluble in water. It gave a molar extinction under the conditions of the assay of 7.22 x 106. A unit of alkaline RNase activity was defined as that activity which produced an absorbance increase at 260 nm equivalent to the production of 1 pmol
Within- and between-batch precision was assessed both by replicate assays and by the technique of randomized pairs. The results are shown in Table 1. Using randomized pairs a higher within-batch coefficient of variation was obtained probably due to the fact that specimen to specimen variations, e.g., protein and lipid composition, are included whereas replicate assays of the same specimen exclude these variations. Between-batch, replicate assays over a longer time interval give better indication of the routine performance of the method. Normal Values
Alkaline RNase activity was assayed in cord blood, plasma from infants and children, and serum from adults all of whom were normal and on average diets; data are shown in Table 2. There is an initial increase TABLE WITHIN-
AND
1
BETWEEN-BATCH
OF THE ASSAY ALKALINE
PRECISION
FOR HUMAN SERUM RNase ACTIVITY CO&Mean activity (units/liter)
SD
cient of variation (96)
Within-batch precision By replicate assays By randomized pairs
585.4 505.8
18.4 24.8
3.15 4.90
Between-batch precision By replicate assays By randomized pairs
326.6 504.8
19.8 21.2
6.05 5.38
238
P. H. SCOTT TABLE AGE RELATED
NORMAL
VALUES
2
FOR HUMAN
Mean activity (units/liter)
SERUM
ALKALINE
RNase
ACTIVITY
SD
Range
n
P
1
BIOCHEMISTRY
A Method
loI&
233-239
(1979)
for the Determination Activity
of Alkaline Ribonuclease in Human Serum
(EC 3.1.4.22)
P. H. SCOTT Biochemistry
Department, Selly Oak Hospital, Birmingham
29, United Kingdom
Received January 11, 1979 The development and evaluation of a method for the determination of alkaline ribonuclease (RNase; EC 3.1.4.22) activity in human serum is described. Transfer RNA at a concentration of 4.0 g/liter is used as substrate. The effects of monovalent and divalent cations, phosphate, and EDTA have been studied. The method is standardized with cytidine, and appropriate units of activity are defined. It is linear up to an activity of 800 units/liter. The within batch coefficient of variation was 4.%, and between batch it was 6.0%. Age-related normal values of serum alkaline RNase activity in infants, children, and adults are given.
Since the first demonstration of RNase activity in blood by Zittle and Reading (I), there have been numerous studies involving the determination of the activity of the serum enzyme in normal and pathological conditions. The increase in RNase with uremia is well known (2,3); however, its association with carcinoma is less well established. Both normal (45) and elevated enzyme activities (6,7) have been claimed. Whether these disparate observations result from histological dissimilarities in the tumors as suggested by Chretien et al. (8) or from methodological inadequacies remains unresolved. Recently it has been confirmed that serum alkaline RNase activity can be related to nitrogen balance (9) and thereby becomes a valuable indicator of nutritional status, being considerably increased in malnutrition (10,ll). The potential of human serum alkaline RNase assays, particularly with respect to more delicate changes in activity, remains to be explored. It has been shown that in normal low-birth-weight babies the serum enzyme correlates well with nitrogen retention (12), and that differences in enzyme activity can be related to the nature of the dietary protein during early postnatal 233
life ( 13). Similarly, during pregnancy racial differences in human serum alkaline RNase levels have been found which were probably nutritional in origin (14) and which may allow identification of pregnancies which result in light-for-gestational-age babies (15). For studies such as these, a sensitive and optimized assay procedure is vitally important. Though turbidimetric (5,16) and colorimetric (17) methods have been developed, serum alkaline RNase activity is usually determined spectrophotometrically by measuring the absorbance of the short-chain oligonucleotides produced, after differential precipitation of substrate and products. Most use has been made of the methods devised by Ambellan and Hollander (18) and by Roth (19). These were, however, developed on and for use with tissue extracts and it is unlikely that they provide a valid optimized assay of human serum enzyme activity. Even among allegedly serum assays there exist many anomalies. The concentration of the RNA substrate used varies from 0.83 (20) to 13.3 g/liter (21); the optimum pH for the human serum enzyme ranges from 6.5 (3) to 8.5 (8); a variety of different ions have been used for activation of the enzyme. 0003-2697/79/180233-07$02.00/O Copyright 8 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
234
P. H. SCOTT
The mode of calibration and derivation of units of activity used in many of the methods is unacceptable. The simplest form relates enzyme activity to, and expresses it in terms of, the absorbance difference produced in the assay (820). Such an arbitrary system negates any form of true calibration, being dependent more on the optical system of the spectrophotometer and the sensitivity of the photodetector. The stability and comparative ease of purification of RNase has facilitated its use as a standard material for calibration of all types of assays (2,5,9,10,21). In most cases bovine pancreatic RNase has been used, but differences between bovine and human enzyme (22) render this material unsuitable. It is also unacceptable to express in mass terms (pg RNase/liter) that which is measured as an activity. Some method of calibration was therefore required which was more consistent with modern trends in enzyme analysis (23). As a consequence of the inadequacies of the methods currently available, it was opportune to reexamine particularly those characteristics of human serum alkaline RNase which are relevant to its assay, and from that to devise a method specifically suited to the human serum enzyme. METHOD
acetate in 70 ml distilled water; this was adjusted to pH 5.5 and made up to 100 ml with water. For use 12 ml of solution A was combined with 1 ml of solution B and with 9 ml distilled water. Cytidine calibration solution, 30 mmoY liter was prepared by dissolving 0.0729 g cytidine in distilled water to a volume of 10 ml. Procedure Into 3-ml polypropylene test tubes was pipetted 300 ~1 of buffered substrate, and into an equivalent number of similar test tubes was pipetted 300 ~1 of Tris-HCl buffer. For each batch of determinations enough tubes were set up for the tests, a calibration solution, a quality-control serum, and a substrate blank. To each tube was added 30 ~1 of 2.0 mol/liter sodium chloride solution. The tubes were then placed in a 37°C constant-temperature water bath for 5 min, and to each pair of tubes was added 10 ~1 of serum, test or control, or calibrant. After incubation for 60 min the reaction was terminated by addition of 2.5 ml of precipitating reagent. The solutions were chilled at -20°C for 10 min and were centrifuged at 3000g for 10 min. The supematants were measured spectrophotometrically against water at 260 nm in a lo-mm path-length silica cell.
Reagents
Calculation
Buffered substrate was prepared by dissolving 113 mg transfer RNA from bakers’ yeast (Boehringer Corp. (London) Ltd.) in 20 ml 0.04 mol/liter Tris-HCl buffer, pH 7.4, and then the solution was made up to 25 ml with buffer. Sodium chloride, 2.0 moYliter in distilled water. Precipitation reagent was prepared as two stock solutions. Solution A was prepared by dissolving 0.07 g lanthanum nitrate in 5 ml distilled water, the solution than being made up to 100 ml with anhydrous ethanol. Solution B was prepared by dissolving 5.58 g magnesium acetate and 8.20 g sodium
AOD26o = test ODzsO - (test blank OD260 + substrate blank OD,,,). Alkaline RNase activity (units/liter) =
of Results
AODm calibrant OD260 X
EVALUATION
[calibrant
(mmoYliter)] 0.06
OF ASSAY CONDITIONS
Choice of Substrate and Substrate Concentration Though there were advantages to be gained from the use of synthetic substrates,
DETERMINATION
OF ALKALINE
in general their inaccessibility precluded them. Polycytidylic acid has been employed (3,24) but, although it gives a highly sensitive assay, not all the isoenzymes of human serum alkaline RNase are equallly effective against the secondary phosphate esters of cytidine 3’-phosphate (25). Also, in the United Kingdom, the high cost of this substrate would prohibit its wide routine use. It was therefore decided to use naturally occurring yeast RNA, but to select the transfer RNA fraction which gave better assay precision (26). Two yeast transfer RNA products (tRNA(a) from Boehringer Corp. (London) Ltd., and tRNA(b) from Sigma Chemical Co., Ltd.) and one transfer RNA from Escherichia coli (tRNA(c) from B.D.H. Chemicals Ltd.) were examined. Both gave low blank values though only tRNA(a) conformed to Roth’s rigorous criteria of acceptability (19). The relationship of enzyme activity to substrate concentration was studied for each of the tRNAs over the range O-4.0 g/liter. The tRNA(b) substrate failed to give anything approaching a maximum velocity for the reaction. The Michaelis constants given by substrates tRNA(a) and tRNA(c), respectively, were 0.92 and 0.43 g/liter, demonstrating the low substrate affinity characteristic of enzymes which act on high molecular weight substrates. All substrates after reaction and precipitation gave acid-soluble oligonucleotide products of reaction with absorption maxima at 260 nm. The tRNA(a) substrate was chosen for use in the assay, at a concentration of 4.0 g/liter.
RNase IN HUMAN
235
SERUM
suggested that both acid (pH 6.5) and alkaline (pH 7.6) RNases exist. This was investigated over the pH range 6.2 to 7.8 using (i) 0.04 and 0.08 mol/liter imidazole (for pH 6.2 to 7.0) and Tris (for pH 7.05 to 7.8) buffers, and (ii) 0.055 mol/liter phosphate-borate buffer (equivalent in this assay to that used by Reddi (3,22)). The results are shown in Fig. 1. With the imidazole/Tris buffer combination two definite peaks of RNase activity, with optima at pH 7.06 and 7.4, were observed. The latter was little affected by increase in buffer concentration provided that sodium chloride was included in the assay solution; if this was absent then enzyme activity continued to increase with increasing buffer concentration. With the more acidic activity there was an increase associated with the higher buffer concentration. The phosphate-borate buffer, on the other hand, gave a single broad peak of activity which was a maximum at pH 6.86. As has been demonstrated (22) phosphate
500 ALKALINE RNASE ACTIVIN 400
UNITS/L
Effect of pH and Buffer Concentration There is some disagreement over the optimum of pH of human serum RNase activity. In tissue extracts both acid and alkaline RNases have been reported. In human serum Reddi (22) has indicated that only an acid RNase optimum at pH 6.5 is present, while Coombes et al. (27) have
7.0
pH
a"
FIG. 1. Effect of pH on the activity of human serum alkaline RNase using 0.04 moYliter imidazole/Tris (0 l ), 0.08 mol/liter imidazole/Tris (0 - - - 0), and 0.055 moYliter phosphate-borate (A A) buffers.
236
P. H. SCOTT
activates serum RNase activity. This has, however, been shown to be accompanied in the case of bovine pancreatic RNase with a shift in the pH optimum to the acid (28). The results obtained here indicate that this also happens with the human serum acid RNase, but that the alkaline RNase retains its higher pH though this becomes obscured by the acid RNase which undergoes greater phosphate activation. Because of this it was not thought appropriate to use phosphate buffer, or to include phosphate ion, in the assay procedure for human serum alkaline RNase. Tris-HCl buffer, pH 7.4 and at a concentration of 0.04 mol/liter was chosen. Effect of Monovalent and EDTA
and Divalent
Cations
A variety of cations, and EDTA, have been included in the reaction solutions for the assay of alkaline RNase activity but there has not yet been a comparative study of their effects. The monovalent cations lithium, sodium potassium, and ammonium (all with chloride as anion) were examined at concentrations from 0 to 350 mmollliter, and the divalent cations magnesium, calium, and cobalt (again with chloride as anion) were studied at concentrations from 0 to 53
500 400 ALKALINE 300 RNA% 200 ACTIVIN
. 100
UN!TS/L ILml
200
300
20
40
60
CATION CONCEhTRATIONW+YIOL/L
FIG. 2. Effects of the monovalent cations Na+ (X-X), K+ (mn ), Li+ (Ol ), and NH,+ (A - - - A), and the divalent cations Cazf (0 0), Mgz+ (U Cl), and Co*+ (A A) on human serum alkaline RNase activity.
mmol/liter. The results are shown in Fig. 2. All the cations showed an optimal concentration for maximum enzyme activity. The greatest activation was by sodium and potassium, and the former was chosen for inclusion in the assay at a concentration of 175 mmol/liter. Both Farkas (29) and Houck and Berman (5) had also added sodium but they used it at the lower concentration of 130 mmol/liter. There was no advantage in including a divalent cation as Biswas and Hindocha (20) have done. Addition of magnesium in the presence of 175 mmol/ liter sodium produced an immediate and progressive decrease in enzyme activity, probably due to competitive inhibition. The effect of EDTA was studied over the concentration range O-O.9 mmol/liter, using Tris salt to dissolve the EDTA so as to avoid addition of sodium or potassium. It was shown that EDTA has no effect on human serum alkaline RNase activity and its inclusion in the assay, as proposed by Shenkin et al. (26), was unnecessary. Calibration
of the Assay
Two factors determine the nature of the reaction products which are measured as an absorbance at 260 nm. The first is the specificity of the enzyme itself and the second is the selectivity of the precipitating reagent. Human serum alkaline RNase acts predominantly on the cytidine 3’-phosphate linkages, though certain of the isoenzymes show activity toward uridine 3’-phosphate linkages (25). Alkaline RNase activities of a series of serum specimens were redetermined and compared with the activities measured using polynucleotides as substrates in place of tRNA. With polyadenylic and polyguanylic acids there was no correlation, but a highly significant correlation was obtained with both polycytidylic and polyuridylic acids (Fig. 3). The activity shown with polycytidylic acid was much greater than that shown toward polyuridylic acid. Of the oligonucleotide fragments
DETERMINATION
OF ALKALINE
RNase IN HUMAN
cytidine per liter, stated conditions.
237
SERUM
per minute,
under the
Linearity
Using both a serum specimen with a high alkaline RNase activity and a purified preparation of the human serum enzyme, it was shown that the method was linear up to 800 units/liter. Sera with activities greater than this were repeated using a 5~1 sample. .
ACTIVITY
0.3
Precision
.
-
AOD'EONM
0.6
0.7 ALKALINE
0.8
0.9
1.0
RNASE ACTIVITY AODXONM
FIG. 3. The correlation of human serum alkaline RNase activity with the activity measured using polycytidylic acid (&) (r = 0.877, P < 0.001) and using polyuridylic acid (A) (r = 0.919, P < 0.001). Enzyme activity is expressed as AOD,,,.
resulting from hydrolysis of tRNA, the majority are therefore likely to be short sequences with cytidine as the terminal base. After hydrolysis, the oligonucleotide fragments are selectively precipitated so that only the acid-soluble products of up to five or six nucleotides in length remain in solution (30). The solubility of the uridylic acidcontaining fragments is less than that of the cytidylic acid-containing ones, so that precipitation favors the former. Cytidine 3’-phosphate was examined as a material for calibration of the assay but was found to be unsuitable. Cytidine however was shown to be a good substitute, being highly soluble in water. It gave a molar extinction under the conditions of the assay of 7.22 x 106. A unit of alkaline RNase activity was defined as that activity which produced an absorbance increase at 260 nm equivalent to the production of 1 pmol
Within- and between-batch precision was assessed both by replicate assays and by the technique of randomized pairs. The results are shown in Table 1. Using randomized pairs a higher within-batch coefficient of variation was obtained probably due to the fact that specimen to specimen variations, e.g., protein and lipid composition, are included whereas replicate assays of the same specimen exclude these variations. Between-batch, replicate assays over a longer time interval give better indication of the routine performance of the method. Normal Values
Alkaline RNase activity was assayed in cord blood, plasma from infants and children, and serum from adults all of whom were normal and on average diets; data are shown in Table 2. There is an initial increase TABLE WITHIN-
AND
1
BETWEEN-BATCH
OF THE ASSAY ALKALINE
PRECISION
FOR HUMAN SERUM RNase ACTIVITY CO&Mean activity (units/liter)
SD
cient of variation (96)
Within-batch precision By replicate assays By randomized pairs
585.4 505.8
18.4 24.8
3.15 4.90
Between-batch precision By replicate assays By randomized pairs
326.6 504.8
19.8 21.2
6.05 5.38
238
P. H. SCOTT TABLE AGE RELATED
NORMAL
VALUES
2
FOR HUMAN
Mean activity (units/liter)
SERUM
ALKALINE
RNase
ACTIVITY
SD
Range
n
P
1
