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A simple fast micromethod for measuring enzyme activities which release tritium as tritium water
ANALYTICAL
BIOCHEMISTRY
161,529-532
(1987)
A Simple Fast Micromethod for Measuring Enzyme Activities Which Release Tritium as Tritium Water* WALTER L. HUGHES Medical Department, Brookhaven National Laboratory, Upton. New York 119 73 Received November 3, 1986 The entire procedure is carried out in a counting vial by mixing the reagents as a 20- to 30-1.11 drop in the cap of a counting vial, incubating, quenching the reaction, and then distilling the tritium water produced into the chilled vial, in which it is assayed after the addition of scintillation solvent and a clean cap. The application of this technique to the analysis of serum transaminases is described. 0 1987 Academic press, Inc. KEY WORDS: tritium; HTO; serum transaminase.
Most procedures for measuring the rates of enzymatic reactions require a succession of manipulative steps. These may include transfer of measured amounts of several components of the reaction mixture to a reaction vessel, temperature equilibration, addition of a measured volume of the sample to be analyzed, precise timing of the duration of the reaction, and halting the reaction at an appropriate interval by a quenching reagent followed by the transfer of a measured volume of the reaction products to a cell for the measurement of a product of the reaction. These multiple steps require the efforts of a technician, limiting the number of assays which can be performed and multiplying the opportunities for procedural errors, although modern automated procedures using Technicon autoanalyzers can largely eliminate these difficulties. We have developed a simple alternative technique which can eliminate all transfers when the product of the reaction is tritium water (HTO)’ and applied it to the assay of serum transaminases.
* This paper is dedicated to the memory of Nathan 0. Kaplan. ’ Abbreviation used: HTO, tritium water.
MATERIALS
AND METHODS
A metal cap with a tight-fitting rubber gasket and a 20-ml counting vial are used for the reaction chamber. Glass counting vials with plastic screw caps (24-mm diameter) were obtained from Wheaton Glass Co.* Metal caps to fit these vials were obtained from Ferdinand Gutmann and Co. (Brooklyn, NY) through the courtesy of Mr. Edward Primeau. The cardboard liners were removed and replaced with rubber gaskets cut as rings, 23 mm in diameter with a l-cm hole, from sheets of 2-mm-thick neoprene rubber. Before insertion, the inside of the cap was coated with a silicon polymer (Krylon spray). Radioactive L-aspartic acid and L-alanine, labeled on the 2,3 positions with tritium (20-50 Ci/mmol), were obtained from Amersham. Serum or plasma samples were obtained from mice or were control standards for clinical assay from Hyland Diagnostics. Beckman liquid scintillation counters and counting solvents were used. The incubating oven with mechanical convection controlled temperatures to +_0.5”C and the “hot plate” was a constant-tempera’ Wheaton 986562 vials with 22 mm caps are also sold, but matching metal caps may be hard to find.
529
0003-2697/87
$3.00
Copyright 0 1987 by Academic Press, Inc. All rights of reproduction in any form reserved
530
WALTER
ture heating block with a range of 5-l 30°C. The block was inverted in its holder to provide a continuous heating surface and a thermometer well was drilled into it. Labeled Laspartic acid and L-alanine may require purification before use, since these produce volatile radioactive products in addition to HTO during storage. Conventional paper chromatography using butanol-acetic acidwater has proven adequate. This permits obtaining a control value (no serum blank) of less than 1% of the total count. The preferred method is as follows: The vial and cap are prewarmed in a constanttemperature oven, which contains a metal plate on which the vial can be placed inverted for rapid temperature equilibration of the reaction mixture. The mixture is added to the cap in two parts: 1O-p*1portions of substrate and enzyme. The cap and vial are rapidly reassembled and placed in the oven on the metal plate. After a predetermined time, the reaction is terminated by transferring the vial, still inverted, to a hot plate thermostated at 85-95°C and a piece of dry ice is placed on the vial to condense the water. In 5 min when evaporation is complete, the vial is righted and allowed to stand at room temperature for 1 min. The reaction cap is removed, counting solvent is added, and a clean cap is screwed on for counting. In order to obtain the highest precision in an assay, several requirements must be met: (1) There must be no escape of radioactivity during the process. It is well to test the seal between cap and vial by immersing the sealed vial in hot water for a few seconds, observing whether any bubbles escape as the vial warms. A more critical test is to carry out a complete run using 20 ~1 HTO in the cap and to determine the recovery of counts. This should exceed 90%. (2) At 50°C the vapor pressure of HZ0 is 93 mm of mercury so that about 1 ~1 of the sample will be in the gas phase within a 20-ml counting vial. This will result in a corresponding decrease in the volume of the re-
L. HUGHES
action mixture during incubation with a corresponding increase in the reaction rate. However, for assays comparing enzyme levels with a standard, the ratios of activity should be constant. (3) Temperature control must be adequate, not only in the incubating oven but also on the hot plate. To ensure this, a copper block with a thermometer well is used for the hot plate. Metal caps are used to provide rapid temperature equilibrium. These are coated with a plastic film such as a silicon spray (Krylon) to prevent chemical interaction with the sample. A drop of serum albumin solution placed on the cap becomes opaque within 15 s after the cap is placed on the hot plate at 85°C. This indicates that temperature inactivation must occur within a fraction of a minute after placing a reaction vial on the hot plate. Care must be taken to avoid too high a temperature as splattering of the sample may occur, transferring nonvolatile activity to the vial walls. This has been observed to occur when the hot plate temperature slightly exceeds 100°C. (4) The procedure for loading the vial should be practiced to develop a standard minimum time for removing the vial from the oven, loading it, and returning it. Our procedure is to store vials and caps in the oven together with an insulating holder for a cap (a large rubber stopper with a well which will hold a cap snugly). Aliquots of sample and substrate are drawn into the disposable plastic tips of dispensing pipettes. Then the oven door is opened, a cap inserted into its holder and removed, and the door promptly closed. The substrate and then the sample are pipetted on the center of the cap and mixed by running the mixture up and down in the plastic tip (time 0). A vial from the oven is screwed rapidly and tightly to the cap and the assembly is returned to stand on a metal plate in the oven. To terminate the reaction the vial is removed and placed on the 90°C hot plate and a piece of dry ice is immediately placed on the vial. Reaction time is recorded as from time 0 to placement
ASSAY
FOR
MEASURING
on the hot plate. During the evaporation, vapors can be seen swirling in the vial. One minute after evaporation is complete (3-5 min), the vial is removed and righted to allow the hot vapors to settle to the cold bottom of the vial, then the cap is removed, counting solvent is added, and a clean plastic cap is screwed on for scintillation counting. RESULTS
This technique has been applied to the radioassay of serum transaminase described by Shuster et al. (1). (For a discussion of the enzyme see Velick and Vavra (2).) They measured the tritiated water formed by removing the unreacted aspartic acid on an anion-exchange column, but suggested that the separation might be accomplished by evaporating the water in a Thunberg tube. They stopped the reaction by cooling the reaction mixture, whereas we stop it by heat inactivation. We find it unnecessary to add carrier aspartic acid and prefer a lower concentration of ketoglutaric acid (0.001 M vs their 0.1 M), which is dissolved in 0.1 M phosphate or Tris buffer at pH 7.0. This buffer is preferred if pyridoxal phosphate is to be added to activate any apo-enzyme present (3). However, the coenzyme can deaminate amino acids nonenzymatically (4). Therefore, if added, suitable controls should be included in testing for this possibility. Taking advantage of the heat stability of transaminases, we prefer to react at 50°C to speed up the reaction. Table 1 shows the results of a typical assay. Two commercially available control sera used as clinical chemistry standards were assayed at different dilutions and reaction times. Control 1 was reported to have a high normal level of 37 IU. Control 2 was definitely, but moderately, elevated with 103 IU of activity. The data in Table I show a linear increase with time of incubation. Even 10 min of incubation could readily differentiate between these two samples. Elevated serum aspartate transaminases are indicative of
ENZYME
531
ACTIVITIES TABLE
1
RELEASEOFVOLATILETRITIUM FROM[‘H]ASPARTATE BY STANDARD HUMAN TEST SERA ONINCUBATIONAT 31°C Net cpm’ at incubation (min)
Control serum I Control serum 2 I + I dilution I + 3 dilution
20
40
60
350
650
950
505
950 535
810
time
330
%* 0.1 I
7000
0.16 0.09
* The blank (no enzyme) of 70 cpm has been subtracted. The sera were diluted with f vol of 0. I M phosphate buffer. pH 7.0, containing IO-’ M a-ketoglutaric acid and IO-PI aliquots incubated with IO ~1 of [3H]L-aspartate (15,000 cpm) in isotonic sodium chloride. * Percentage of total counts released per minute.
gross tissue damage as in hepatitis and in coronary occlusion. Like Shuster we have used this technique in assaying hepatotoxins and find it well adapted for studying mice. Repeated 209~1 samples of blood can be drawn from the tail of a single mouse, making it possible to follow the time course of injury from breathing carbon tetrachloride vapors. Mouse blood contains higher levels of enzymes so that dilutions of the plasma can be used. The method is also easily adapted to assaying mammalian tissue cultures, 1O4 cells being adequate for an assay. DISCUSSION
This simplified method of enzyme analysis should prove useful in a variety of applications. It does not require the purchase and maintenance of complicated equipment such as the autoanalyzer and should be relatively free of operator error. The amount of tritium required for analysis is approximately 1 nCi, so that up to 10,000 analyses could be performed with 10 GCi of ‘H. Serum transaminase levels are widely used as indicators of gross tissue damage, which results from coronary occlusions or from liver injury due to hepatitis (5) or chemicals
532
WALTER
( 1). This technique is particularly well adapted for these serum assays. Unlike current procedures, it requires no accessory enzymes with the resulting kinetic complications of coupled reactions. It is unaffected by pigment or turbidity and can in fact be performed on whole blood for the analysis of alanine transaminase, which is absent from red cells. While not applicable for the determination of aspartate transaminase on whole blood, simultaneous assay of separated cells and plasma might provide useful additional data. As we have found, it is a very convenient way of following changes in blood transaminases due to hepatotoxic chemicals. Complete analysis of a sample by one technician is possible within an hour, permitting on-line data not only in the laboratory but also at the bedside.3 This should prove useful in following the progression of coronary occlusions and the testing of blood donors (5). Elevated transaminase levels may also occur in heavy drinkers (6) and might serve as a warning of impending cirrhosis. Application to other enzyme systems will generally require the synthesis of suitably labeled substrates, although the present substrates could be used in measuring L-amino oxidases and substrates for D-amino acid oxidases could be readily produced. Other oxidases such as glucose oxidase and tryptophan oxidase also appear to be good candidates. However, it should be kept in mind that sometimes hydrogen is transferred to the cosubstrate, as in the dehydrogenases (8), from which it must be released by a second enzy3 An alternate rapid technique uses membrane electrodes, but this procedure also requires secondary enzymes (7).
L. HUGHES
matic reaction. Another possible group of enzymes would include decarboxylases (e.g., tyrosine decarboxylase (3)), in which CO* released from a carboxyl group labeled with 14C could be volatilized in the reaction chamber and trapped in an alkaline film on the inner surface of the vial. ACKNOWLEDGMENTS This paper is dedicated to the memory of Nathan Kaplan. We had a long and stimulating relationship, which began when I joined the McCollum Pratt Institute in 1953. Nate had a flair for developing simple, but elegant techniques and would have enjoyed this report. This research was supported in part by Grant ES03302-OlAl from the National Institutes of Health and research facilities were provided by Brookhaven National Laboratory, Associated Universities, Inc., under Contract DE-AC02-76CH00016 with the U.S. Department of Energy. Accordingly, the U.S. Govemment retains a nonexclusive, royalty-free license to pub lish or reproduce the published form of this contribution, or allow others to do so, for U.S. Government purposes. The excellent secretarial assistance of Ms. Bemice Armstrong is also appreciated.
REFERENCES 1. Shuster, L., Bates, H., and Hirsch, C. A. (1978) Anal. Biochem. 86,648-654. 2. Velick, S. F., and Vavra, J. (1963) in The Enzymes, Vol. 6, pp. 2 19-246, Academic Press, New York. 3. Westerhuis, L. W. J. J. M., and Hafkenscheid, J. C. M. (1983) Clin. Chem. 29,789-792. 4. Westheimer, F. H. (1959) Enzyme Models, The Enzymes, Vol. 1, pp. 250-304, Academic Press, New York. 5. Silverstein, M. D., Mulley, A. G., and Dienstag, J. L. (1984) J. Amer. Med. Assoc. 252, 2839-2845. 6. Whitehead, T. P., Clarke, C. A., and Whitfield, A. G. W. (1978) Lance& May 6,978-98 1. 7. Kihara, K., Yasukawa, E., Hayashi, M., and Hirose, S. (1984) Anal. Chem. 56, 1876-1880; Anal. Chim. Acta 159,8 l-86. 8. Vennesland, and Westheimer (1954) in Mechanics of Enzymatic Action, pp. 357-388, Academic Press, New York.
BIOCHEMISTRY
161,529-532
(1987)
A Simple Fast Micromethod for Measuring Enzyme Activities Which Release Tritium as Tritium Water* WALTER L. HUGHES Medical Department, Brookhaven National Laboratory, Upton. New York 119 73 Received November 3, 1986 The entire procedure is carried out in a counting vial by mixing the reagents as a 20- to 30-1.11 drop in the cap of a counting vial, incubating, quenching the reaction, and then distilling the tritium water produced into the chilled vial, in which it is assayed after the addition of scintillation solvent and a clean cap. The application of this technique to the analysis of serum transaminases is described. 0 1987 Academic press, Inc. KEY WORDS: tritium; HTO; serum transaminase.
Most procedures for measuring the rates of enzymatic reactions require a succession of manipulative steps. These may include transfer of measured amounts of several components of the reaction mixture to a reaction vessel, temperature equilibration, addition of a measured volume of the sample to be analyzed, precise timing of the duration of the reaction, and halting the reaction at an appropriate interval by a quenching reagent followed by the transfer of a measured volume of the reaction products to a cell for the measurement of a product of the reaction. These multiple steps require the efforts of a technician, limiting the number of assays which can be performed and multiplying the opportunities for procedural errors, although modern automated procedures using Technicon autoanalyzers can largely eliminate these difficulties. We have developed a simple alternative technique which can eliminate all transfers when the product of the reaction is tritium water (HTO)’ and applied it to the assay of serum transaminases.
* This paper is dedicated to the memory of Nathan 0. Kaplan. ’ Abbreviation used: HTO, tritium water.
MATERIALS
AND METHODS
A metal cap with a tight-fitting rubber gasket and a 20-ml counting vial are used for the reaction chamber. Glass counting vials with plastic screw caps (24-mm diameter) were obtained from Wheaton Glass Co.* Metal caps to fit these vials were obtained from Ferdinand Gutmann and Co. (Brooklyn, NY) through the courtesy of Mr. Edward Primeau. The cardboard liners were removed and replaced with rubber gaskets cut as rings, 23 mm in diameter with a l-cm hole, from sheets of 2-mm-thick neoprene rubber. Before insertion, the inside of the cap was coated with a silicon polymer (Krylon spray). Radioactive L-aspartic acid and L-alanine, labeled on the 2,3 positions with tritium (20-50 Ci/mmol), were obtained from Amersham. Serum or plasma samples were obtained from mice or were control standards for clinical assay from Hyland Diagnostics. Beckman liquid scintillation counters and counting solvents were used. The incubating oven with mechanical convection controlled temperatures to +_0.5”C and the “hot plate” was a constant-tempera’ Wheaton 986562 vials with 22 mm caps are also sold, but matching metal caps may be hard to find.
529
0003-2697/87
$3.00
Copyright 0 1987 by Academic Press, Inc. All rights of reproduction in any form reserved
530
WALTER
ture heating block with a range of 5-l 30°C. The block was inverted in its holder to provide a continuous heating surface and a thermometer well was drilled into it. Labeled Laspartic acid and L-alanine may require purification before use, since these produce volatile radioactive products in addition to HTO during storage. Conventional paper chromatography using butanol-acetic acidwater has proven adequate. This permits obtaining a control value (no serum blank) of less than 1% of the total count. The preferred method is as follows: The vial and cap are prewarmed in a constanttemperature oven, which contains a metal plate on which the vial can be placed inverted for rapid temperature equilibration of the reaction mixture. The mixture is added to the cap in two parts: 1O-p*1portions of substrate and enzyme. The cap and vial are rapidly reassembled and placed in the oven on the metal plate. After a predetermined time, the reaction is terminated by transferring the vial, still inverted, to a hot plate thermostated at 85-95°C and a piece of dry ice is placed on the vial to condense the water. In 5 min when evaporation is complete, the vial is righted and allowed to stand at room temperature for 1 min. The reaction cap is removed, counting solvent is added, and a clean cap is screwed on for counting. In order to obtain the highest precision in an assay, several requirements must be met: (1) There must be no escape of radioactivity during the process. It is well to test the seal between cap and vial by immersing the sealed vial in hot water for a few seconds, observing whether any bubbles escape as the vial warms. A more critical test is to carry out a complete run using 20 ~1 HTO in the cap and to determine the recovery of counts. This should exceed 90%. (2) At 50°C the vapor pressure of HZ0 is 93 mm of mercury so that about 1 ~1 of the sample will be in the gas phase within a 20-ml counting vial. This will result in a corresponding decrease in the volume of the re-
L. HUGHES
action mixture during incubation with a corresponding increase in the reaction rate. However, for assays comparing enzyme levels with a standard, the ratios of activity should be constant. (3) Temperature control must be adequate, not only in the incubating oven but also on the hot plate. To ensure this, a copper block with a thermometer well is used for the hot plate. Metal caps are used to provide rapid temperature equilibrium. These are coated with a plastic film such as a silicon spray (Krylon) to prevent chemical interaction with the sample. A drop of serum albumin solution placed on the cap becomes opaque within 15 s after the cap is placed on the hot plate at 85°C. This indicates that temperature inactivation must occur within a fraction of a minute after placing a reaction vial on the hot plate. Care must be taken to avoid too high a temperature as splattering of the sample may occur, transferring nonvolatile activity to the vial walls. This has been observed to occur when the hot plate temperature slightly exceeds 100°C. (4) The procedure for loading the vial should be practiced to develop a standard minimum time for removing the vial from the oven, loading it, and returning it. Our procedure is to store vials and caps in the oven together with an insulating holder for a cap (a large rubber stopper with a well which will hold a cap snugly). Aliquots of sample and substrate are drawn into the disposable plastic tips of dispensing pipettes. Then the oven door is opened, a cap inserted into its holder and removed, and the door promptly closed. The substrate and then the sample are pipetted on the center of the cap and mixed by running the mixture up and down in the plastic tip (time 0). A vial from the oven is screwed rapidly and tightly to the cap and the assembly is returned to stand on a metal plate in the oven. To terminate the reaction the vial is removed and placed on the 90°C hot plate and a piece of dry ice is immediately placed on the vial. Reaction time is recorded as from time 0 to placement
ASSAY
FOR
MEASURING
on the hot plate. During the evaporation, vapors can be seen swirling in the vial. One minute after evaporation is complete (3-5 min), the vial is removed and righted to allow the hot vapors to settle to the cold bottom of the vial, then the cap is removed, counting solvent is added, and a clean plastic cap is screwed on for scintillation counting. RESULTS
This technique has been applied to the radioassay of serum transaminase described by Shuster et al. (1). (For a discussion of the enzyme see Velick and Vavra (2).) They measured the tritiated water formed by removing the unreacted aspartic acid on an anion-exchange column, but suggested that the separation might be accomplished by evaporating the water in a Thunberg tube. They stopped the reaction by cooling the reaction mixture, whereas we stop it by heat inactivation. We find it unnecessary to add carrier aspartic acid and prefer a lower concentration of ketoglutaric acid (0.001 M vs their 0.1 M), which is dissolved in 0.1 M phosphate or Tris buffer at pH 7.0. This buffer is preferred if pyridoxal phosphate is to be added to activate any apo-enzyme present (3). However, the coenzyme can deaminate amino acids nonenzymatically (4). Therefore, if added, suitable controls should be included in testing for this possibility. Taking advantage of the heat stability of transaminases, we prefer to react at 50°C to speed up the reaction. Table 1 shows the results of a typical assay. Two commercially available control sera used as clinical chemistry standards were assayed at different dilutions and reaction times. Control 1 was reported to have a high normal level of 37 IU. Control 2 was definitely, but moderately, elevated with 103 IU of activity. The data in Table I show a linear increase with time of incubation. Even 10 min of incubation could readily differentiate between these two samples. Elevated serum aspartate transaminases are indicative of
ENZYME
531
ACTIVITIES TABLE
1
RELEASEOFVOLATILETRITIUM FROM[‘H]ASPARTATE BY STANDARD HUMAN TEST SERA ONINCUBATIONAT 31°C Net cpm’ at incubation (min)
Control serum I Control serum 2 I + I dilution I + 3 dilution
20
40
60
350
650
950
505
950 535
810
time
330
%* 0.1 I
7000
0.16 0.09
* The blank (no enzyme) of 70 cpm has been subtracted. The sera were diluted with f vol of 0. I M phosphate buffer. pH 7.0, containing IO-’ M a-ketoglutaric acid and IO-PI aliquots incubated with IO ~1 of [3H]L-aspartate (15,000 cpm) in isotonic sodium chloride. * Percentage of total counts released per minute.
gross tissue damage as in hepatitis and in coronary occlusion. Like Shuster we have used this technique in assaying hepatotoxins and find it well adapted for studying mice. Repeated 209~1 samples of blood can be drawn from the tail of a single mouse, making it possible to follow the time course of injury from breathing carbon tetrachloride vapors. Mouse blood contains higher levels of enzymes so that dilutions of the plasma can be used. The method is also easily adapted to assaying mammalian tissue cultures, 1O4 cells being adequate for an assay. DISCUSSION
This simplified method of enzyme analysis should prove useful in a variety of applications. It does not require the purchase and maintenance of complicated equipment such as the autoanalyzer and should be relatively free of operator error. The amount of tritium required for analysis is approximately 1 nCi, so that up to 10,000 analyses could be performed with 10 GCi of ‘H. Serum transaminase levels are widely used as indicators of gross tissue damage, which results from coronary occlusions or from liver injury due to hepatitis (5) or chemicals
532
WALTER
( 1). This technique is particularly well adapted for these serum assays. Unlike current procedures, it requires no accessory enzymes with the resulting kinetic complications of coupled reactions. It is unaffected by pigment or turbidity and can in fact be performed on whole blood for the analysis of alanine transaminase, which is absent from red cells. While not applicable for the determination of aspartate transaminase on whole blood, simultaneous assay of separated cells and plasma might provide useful additional data. As we have found, it is a very convenient way of following changes in blood transaminases due to hepatotoxic chemicals. Complete analysis of a sample by one technician is possible within an hour, permitting on-line data not only in the laboratory but also at the bedside.3 This should prove useful in following the progression of coronary occlusions and the testing of blood donors (5). Elevated transaminase levels may also occur in heavy drinkers (6) and might serve as a warning of impending cirrhosis. Application to other enzyme systems will generally require the synthesis of suitably labeled substrates, although the present substrates could be used in measuring L-amino oxidases and substrates for D-amino acid oxidases could be readily produced. Other oxidases such as glucose oxidase and tryptophan oxidase also appear to be good candidates. However, it should be kept in mind that sometimes hydrogen is transferred to the cosubstrate, as in the dehydrogenases (8), from which it must be released by a second enzy3 An alternate rapid technique uses membrane electrodes, but this procedure also requires secondary enzymes (7).
L. HUGHES
matic reaction. Another possible group of enzymes would include decarboxylases (e.g., tyrosine decarboxylase (3)), in which CO* released from a carboxyl group labeled with 14C could be volatilized in the reaction chamber and trapped in an alkaline film on the inner surface of the vial. ACKNOWLEDGMENTS This paper is dedicated to the memory of Nathan Kaplan. We had a long and stimulating relationship, which began when I joined the McCollum Pratt Institute in 1953. Nate had a flair for developing simple, but elegant techniques and would have enjoyed this report. This research was supported in part by Grant ES03302-OlAl from the National Institutes of Health and research facilities were provided by Brookhaven National Laboratory, Associated Universities, Inc., under Contract DE-AC02-76CH00016 with the U.S. Department of Energy. Accordingly, the U.S. Govemment retains a nonexclusive, royalty-free license to pub lish or reproduce the published form of this contribution, or allow others to do so, for U.S. Government purposes. The excellent secretarial assistance of Ms. Bemice Armstrong is also appreciated.
REFERENCES 1. Shuster, L., Bates, H., and Hirsch, C. A. (1978) Anal. Biochem. 86,648-654. 2. Velick, S. F., and Vavra, J. (1963) in The Enzymes, Vol. 6, pp. 2 19-246, Academic Press, New York. 3. Westerhuis, L. W. J. J. M., and Hafkenscheid, J. C. M. (1983) Clin. Chem. 29,789-792. 4. Westheimer, F. H. (1959) Enzyme Models, The Enzymes, Vol. 1, pp. 250-304, Academic Press, New York. 5. Silverstein, M. D., Mulley, A. G., and Dienstag, J. L. (1984) J. Amer. Med. Assoc. 252, 2839-2845. 6. Whitehead, T. P., Clarke, C. A., and Whitfield, A. G. W. (1978) Lance& May 6,978-98 1. 7. Kihara, K., Yasukawa, E., Hayashi, M., and Hirose, S. (1984) Anal. Chem. 56, 1876-1880; Anal. Chim. Acta 159,8 l-86. 8. Vennesland, and Westheimer (1954) in Mechanics of Enzymatic Action, pp. 357-388, Academic Press, New York.