Effects of luciferin concentration of the quantitative assay of ATP using crude luciferase preparations

ANALYTICAL

BIOCHEMISTRY

75, lf% 112 (1976)

Effects of Luciferin Concentration on the Quantitative Assay of ATP Using Crude Luciferase Preparations D. M. KARL AND 0. HOLM-HANSEN Institute

of Marine

Resources, Scripps California, San Diego,

Institution La Jolla,

of Oceanography, California 92093

University

of

Received February 23, 1976; accepted May 5, 1976 The reaction kinetics of crude firefly lantern extracts with and without added synthetic luciferin were examined. The addition of exogenous luciferin to the reaction mixture resulted in an apparent increase in net light emission per unit of ATP in solution. This additional reactivity (up to 1000-fold) enables the detection of subpicogram levels of ATP. The effects of enzyme preparation, dilution, and aging procedures on the increased sensitivity of the ATP assay, as well as the assay limitations of the crude firefly lantern extracts, are also discussed.

Within recent years, the firefly bioluminescent reaction has employed in a number of laboratory and field studies as a quantitative for adenosine triphosphate (ATP) (1,2). Several detailed reviews recently been published concerning the specificity, kinetics, mechanism of this enzyme-catalyzed reaction (3-5). The postulated of the reaction are E (luciferase) E-LH,-AMP

+ LH2 (luciferin)

+ ATP ZE-LH,-AMP

+ 0, ne~oxyluciferin

been assay have and steps

+ PP, [l]

+ E + CO, + AMP + hv. [2]

PH

When all necessary reactants are present in excess, the in vitro light emission is directly proportional to the concentration of ATP in the reaction mixture. Either the initial rise of the luminescent curve (6,7), the peak height of luminescence (8), or some integrated portion of the subsequent decay curve (9) can be related to samples of standard and unknown ATP concentrations. Although several investigators have utilized purified reagents (lo- 12), the majority of reports in the literature are based upon measurements obtained with crude enzyme preparations. Even though the firefly lantern extracts available from Sigma Chemical Company (FLE-50, FLE-2.50) are widely used, it is often difficult to compare published results due to the variability in both the preparation and aging procedures of the enzyme mixture (see Table 1). For example, the final volume for a 50-mg vial of firefly lantern extract ranges from 5 to 155 ml, representing more than a 100 Copyright All rights

0 1976 by Academic Press, Inc. of reproduction in any form reserved.

(9)

Helm-Hansen and Booth

155.0

Lin and Cohen

” Enzyme aged until ’ Also added apyrase

(24)

background to firefly

50.0

6.0

Rudd and Hamilton

Schram

37.5

Patterson et al. (21)

(34)

5.00

Lyman and DeVincenzo (33)

(32)

5.0

25.0

Lee et a/. (31)

(30)

5.0

(29)

Conklin and MacGregor

Karl and LaRock

25.0

Beutler and Baluda (28)

5.0

12.5

Addanki et al. (27)

Investigator(s)

Total enzyme volume (ml)

HYDRATION

a predetermined

0.1

0.1

1.5

0.25

2.0

0.2

0.5

0.2

0.1

4.0

0.2

Enzyme/assay (ml)

IN ENZYME

cpm reached lantern extract.

1.0

8.33

1.3

10.0

0.32

10.0

2.0

10.0

10.0

2.0

4.0

FLE (mg ml-‘)

VARIATION

1

level.

0.11

0.3

2.0

1.4

100

1 .o-20

40- 200

0.01-1000

0.2-

1.0

0.5-50

0.3-50

0.5-

-

100-800

5-25

ATP standards (ng ml-‘)

PROCEDURES

2.0

1.5

0.4

0.15

4.2

2.0

Total reaction volume (ml)

AND AGING

TABLE

8-10

1. then

0.5-

48-72

3-4

6-7

2.5-4.0

24

1.o

Variable”

O-96

O-1

Aging conditions time (hr)

FOR SIGMA

4

25

25 0

32

4

25

23-25

25

22

4

0

Temperature (“C)

FLE-50

soil ATP

ATP

kinetic

in lake

in marine

Enzyme

kinetic

study

in freshwater

ATP levels lakes

study

in activated

kinetic

ATP levels sludge

Enzyme

ATP levels in crayfish stretch receptor cells

ATP levels ments

ATP levels sediments

sedi-

levels

levels

study

ATP levels in marine bacteria and algae

Desert

Erythrocyte

Enzyme

Application

102

KARL

AND

HOLM-HANSEN

30-fold variation in the luciferin and luciferase concentrations. Furthermore, the treatments of these enzyme preparations, with respect to the temperature and the time of aging, exhibit similar variations. Since a Mg-ATP complex is the actual substrate in the firefly reaction (3), it is advisable to add exogenous Mg 2+ to the enzyme mixture whenever the final volume exceeds that specified by the manufacturer (i.e., 5 ml for the FLE-50, 25 ml for the FLE-250). Although most investigators have assumed that luciferin is present in excess in the crude enzyme preparations, we noticed that the addition of exogenous luciferin to the reaction mixture resulted in a substantial increase in light emission. This paper elucidates the response of FLE-50 enzyme to the addition of exogenous luciferin, evaluates the reaction kinetics and assay limitations of the crude firefly lantern extracts and describes a more sensitive set of reaction conditions. MATERIALS

AND METHODS

The disodium salt of ATP, Tris buffer (pH 7.7), syntheticD(-)-luciferin, and firefly lantern extracts (FLE-50, 50 mg/vial) were all obtained from Sigma Chemical Company (St. Louis, MO.). All other chemicals used in this study were analytical grade reagents. Stock ATP solutions (1 pg ml-‘) were prepared in 0.02 M Tris buffer and stored at -20°C. Working solutions were prepared on the day of each assay via dilution with 0.02 M Tris buffer. A synthetic luciferin solution was prepared to a concentration of 1 mg ml-l in 0.02 M Tris buffer, and stored at -20°C until used. This stock solution was diluted with Tris buffer to a series of concentrations ranging from lo-600 pg ml-‘, immediately prior to use. Since luciferin is unstable in oxygen and light (13), caution should be taken in the preparation and storage procedures. Prior to use, lyophilized firefly lantern extracts were stored desiccated at -20°C. Each vial was reconstituted with 5.0 ml of distilled water. When rehydrated to this volume, the buffered medium (pH 7.4) contained 0.05 M K,HAsO, and 0.02 M MgSO, as described by the manufacturer. Whenever further dilution of the enzyme was required for comparative studies, equal volumes of 0.1 M K,HAsO, (pH 7.4) and 0.04 M MgS04 were added in order to maintain a buffered ionic medium consistent with that described above. For each ATP analysis, 0.5 ml of the appropriate ATP standard was injected into a scintillation vial containing 0.5 ml of the enzyme preparation and 0.5 ml of the experimental luciferin solution. Tris buffer was added to the control vials in order to achieve comparable reaction volumes. The vial was mixed thoroughly for 5 set and inserted into the photometer. Light emission was measured with a commercial ATP photometer (SAI Tech. Co., La Jolla, Ca.). The counts obtained (expressed as counts

EFFECTS OF LUCIFERIN

0 4 8 ATP CONCENTRATION ng ml-l

75 ;IME,

15 0

103

-

75

15 0 TIME, f

(set)

12

ON ATP ASSAY

( sex)

FIG. 1. Kinetics of the firefly bioluminescent reaction as a function of ATP concentration. The ATP standards were injected and the recorder automatically turned on at time = 0 sec. The cpm represent the integrated area under the decay curve between 15-7.5 set, as described in Materials and Methods.

per minute (cpm)) were equivalent to the integrated area under the luminescent decay curve between 15 and 75 set after the start of the reaction. For several experiments, the peak height of luminescence and the subsequent decay kinetics were monitored by interfacing the photometer with an analog recorder. In these latter experiments, the ATP samples were injected using special autopipet fittings (SAI Tech. Co., La Jolla, Ca.) designed to permit mixing of the reagents while the vial is inserted in the counter. RESULTS AND DISCUSSION

Effects of Subsaturating

Luciferin

Concentrations

The bioluminescent reaction mechanism proposed by McElroy and DeLuca (3) indicates that the formation of the luciferyl-adenylate complex (E-LH,-AMP) is the rate limiting reaction for the overall process. If in such a bisubstrate reaction, we add enough of one substrate (LH,) to saturate the system, then the subsequent reaction rate can be described by first order kinetics with respect to the rate limiting substrate (ATP). This linear relationship between quanta of light emitted and ATP consumed is the kinetic basis for the firefly ATP assay (14). When standard ATP solutions are assayed using the firefly enzyme system, a direct relationship is observed between peak height (or integrated area) and ATP concentration (Fig. 1). However, when the concentration of ATP is kept constant, and exogenous luciferin is added to the reaction mixture, there is a great enhancement in light emission (Fig. 2).

104

KARL AND HOLM-HANSEN

,04-

0

100

200

LUCIFERIN

300

400

500

ADDEDpg

FIG. 2. Variation in light emission (expressed as cpm) for a standard ATP concentration as a function of exogenous luciferin. The enzyme preparation was Sigma FLE-50 reconstituted to a total volume of 5 ml with distilled water, and the total ATP concentration in each sample was 25 ng. This enzyme extract was used immediately after hydration.

This suggests that the enzyme preparation is subsaturated with respect to luciferin, and the original assay assumption is no longer satisfied. As the concentration of luciferin is increased to 100 pg, light emission rises to a maximum level 15-20 times greater than that of the control for an ATP concentration of 50 ng ml-‘. At very high luciferin concentrations (> lOO150 pg), there is an inhibition of the light output below this maximum level. It is apparent from these kinetic data that the total light emission is dependent upon the concentration of luciferin in this particular reaction mixture. Since the integral mode of analysis was utilized in all of the comparative studies, the luminescent decay curve for the ATP-firefly mixture was monitored to examine the possibility that the apparent increase in activity might be the result of altered reaction kinetics. Figure 3 shows that both the peak height of luminescence and the integrated area between 15 and 75 set increased with the addition of luciferin to the reaction mixture. Furthermore, the shape of the decay curves resembled those shown for the standard ATP solutions (i.e., Fig. I), indicating that the addition of luciferin did not alter the characteristics of the reaction decay kinetics. Additional experiments have indicated that the time course of the luminescent decay curve is not altered through the addition of exogenous luciferin. Therefore, the addition of luciferin to the reaction mixture allows a greater amount of light to be emitted over a given period of time for a constant ATP concentration. These data could be interpreted in at least two different ways: (1) The addition of luciferin to the enzyme mixture results in an increased quantum yield for the reaction, with respect to ATP (i.e., increase in quanta emitted per ATP molecule hydrolyzed); (2) The addition of luciferin to the

EFFECTS OF LUCIFERIN

ON ATP ASSAY

105

FIG. 3. Kinetics for the firefly bioluminescent reaction when exogenous luciferin is added to a standard ATP concentration. The enzyme preparation was Sigma FLE-50 reconstituted to 5 ml with distilled water, and the total ATP concentration in each sample was 0.5 ng.

reaction mixture results in an increased reactivity of the ATP in solution. Although Seliger and McElroy (15) have reported that the quantum yield of the firefly reaction is unity with respect to luciferin (i.e., 1 quantum emitted per luciferin molecule oxidized), to our knowledge no comparable measurements have been made with respect to ATP hydrolysis. The observation that synthetic LH,-AMP relieves the firefly system of its ATP requirement (3) suggests that only a single molecule of ATP is necessary for the formation of the luciferyl-adenylate reaction complex. If the latter interpretation is correct, then the reaction mixture must be undersaturated with respect to available luciferin in solution. The original ATP assay assumption, that the reaction rate will depend solely upon the ATP concentration, is not in agreement with these activity measurements using exogenous luciferin. Effect of Enzyme Preparation,

Dilution,

and Aging Procedures

A series of experiments was conducted to determine the effect of dilution of the enzyme extract on the sensitivity of the ATP assay. The results of this study are summarized in Fig. 4. When luciferin is added to a reaction mixture containing a low level of ATP (2 ng ml-‘), there is an increase in cpm evident in all four of the enzyme dilutions (FLE-50 reconstituted to 5, 10, 25, and 50 ml). However, when a comparison is made between the total light emission at 0 and 25 pg exogenous luciferin for each enzyme preparation, the endogenous substrate dilution effect is once again evident

106

KARL AND HOLM-HANSEN

t

LUCIFERIN

ADDEDpg

FIG. 4. Increase in light emission (expressed as cpm) for a standard ATP concentration as a function of exogenous luciferin. The total ATP concentration in each sample was 1.0 ng. All four enzyme preparations were prepared as described in Materials and Methods and were aged for 6 hr at 25°C. Symbols: closed circle, FLEJO reconstituted to 5 ml; open circle, FLE-50 reconstituted to 10 ml; closed square, FLE-50 reconstituted to 25 ml; open square, FLEJO reconstituted to 50 ml.

(i.e., cpm,, clp/cpmo FLg= 22.5 for 50 ml, 12.3 for 25 ml, 5.6 for 10 ml, and 4.8 for 5 ml). The increased activity of the lo-ml enzyme preparation, and of the 25-ml preparation at luciferin additions greater than 7 pg, relative to the more concentrated 5-ml extract, might possibly be the result of dilution of the inhibitory end-product, dehydroluciferin. Although oxyluciferin is the primary end-product during luciferin oxidation (16,17), some dehydroluciferin (L) is also formed as a result of the enzymatic process (3). In the presence of luciferase, dehydroluciferin is activated to form the enzymebound dehydroluciferyl-adenylate complex (E-L-AMP), but no light is subsequently emitted (18). Moreover, dehydroluciferin has been shown to be a competitive inhibitor of the firefly bioluminescence reaction (17,19,20). The production of dehydroluciferin in the reaction mixture is a direct result of the endogenous light emission, and is therefore a function of both enzyme concentration and aging time. The longer the enzyme is aged and the more concentrated it is, the greater the formation and accumulation of dehydroluciferin. Therefore, dilution of the firefly lantern extract permits a greater number

EFFECTS

OF LUCIFERIN TABLE

107

ON ATP ASSAY 2

TEMPORAL CHANGESIN cpm FORA STANDARD ATP CONCENTRATION AS A FUNCTION OF EXOGENOUS LLJCIFERIN~ Time (hr) at 25°C 0 0 0 0 0

Luciferin added (a)

Net cpm

Time (hr) at 25°C

Luciferin added 0449

Net cpm

0 5 10 25 50

7.52 x 1.26 x 1.78 x >6.5 x >6.5 x

10” lo” 105 105’ lo5

12 12 I2 12 12

0 5 10 25 50

5.59 9.60 1.39 >6.5 >6.5

x x x x x

101 IO” 105 lo5 IO5

0 5 10 25 50

7.27 1.24 1.65 >6.5 >6.5

x x x x x

10’ 105 IO” lo5 lo5

24 24 24 24 24

0 5 IO 25 50

4.02 x 6.51 x 8.99 x 1.50 x >6.5 x

IO’ 10’ 101 105 IO5

0 5 10 25 50

7.22 x 1.22 x 1.66 x >6.5 x >6.5 x

IO4 loj 105 lo5 lo5

36 36 36 36 36

0 5 10 25 50

-c 6.10 9.00 1.53 >6.5

10” 10” I05 IO5

x x x x

(1The enzyme preparation utilized in this experiment was FLE-50 reconstituted to 5 ml with distilled water and the ATP concentration was 10 ng ml-‘. b All cpm greater than 6.5 x IO5overloaded the electronic capabilities of the photometer at this particular sensitivity level. c Below detectable limits (< 100 cpm).

of assays to be made from a single vial, decreases the concentration of inhibitory end products and reduces the level of endogenous ATP, thereby decreasing the background light emission and increasing the sensitivity of the assay. Temporal

Enzyme Activity

Several investigators have reported a decrease in the activity of the enzyme as a function of time after reconstitution with distilled water (21-23). Our results indicate that this is not the case. An experiment was performed in order to evaluate a variety of temporal changes that occur in the response to added luciferin for a standard ATP concentration. Table 2 indicates that even after 36 hr at 25°C the enzyme preparation can be restored to maximal activity through the addition of exogenous luciferin, demonstrating that luciferase is stable under these conditions. These data also show that for a standard ATP concentration, a greater amount of luciferin must be added to achieve a given activity, as the aging time is

108

KARL AND HOLM-HANSEN 260

i 240-

&-zoo-

b x z 5

160-

l 24 hn

20

LUCIFERIN

24

26

ADDED/q

FIG. 5. Temporal changes in the endogenous background light emission as a function of added luciferin.

increased. This suggests that the enzyme mixture is becoming more undersaturated with respect to luciferin as a function of time. Figure 5 relates the endogenous background light emission (i.e., light level prior to the addition of sample ATP) from this same series of experiments as a function of exogenous luciferin. It is evident that the background light emission decreases as a function of aging time and that after 36 hr of incubation at 25°C the endogenous light emission was essentially independent of luciferin concentration (Fig. 5). These results could be explained either by a decrease in the endogenous ATP concentration or by an increase in the concentration of some inhibitory end-product, or both. ,Sensitivity and Reproducibility

of the Assay

For any given concentration of ATP, the enzymatic activity (cpm/ng of ATP) will be greatest when the system is saturated with luciferin. It is apparent from Fig. 6 that the gross enzymatic activity (response due to added ATP plus endogenous ATP) increases more than 30-fold as the luciferin saturation level is achieved. As luciferin is added to the reaction mixture, there is a concomitant increase in the background light emission (Fig. 6), primarily as a result of the ATP in the crude enzyme preparation. As discussed previously, this increase in background activity is a direct function of enzyme dilution and aging. The most important consideration in determining assay sensitivity is to examine the net cpm, or activity above background light emission. Figure 6 indicates that the net cpm increase with luciferin saturation to a level three orders of magnitude greater than the control sample. The maximum net enzymatic activity for this ATP sample (5 pg) was 2.5 x 104 cpm at an exogenous luciferin addition of 250 ,ug. These results indicate that the crude Sigma firefly lantern extract

EFFECTS

OF LUCIFERIN

~OOoio LUCIFERIN

ON ATP ASSAY

109

ADDEDpg

FIG. 6. Increase in light emission (expressed as cpm) for a standard ATP concentration as a function of exogenous luciferin. The enzyme preparation was Sigma FLE-50 reconstituted to 5 ml with distilled water, and the ATP concentration in each sample was 5.0 pg. Symbols: triangle, gross cpm; square, background cpm; circle, net cpm.

(FLE-50), when used under these conditions, is capable of detecting ATP levels in the subpicogram range. When working with low pulse amplitudes resulting from single photon emissions as in the bioluminescent reaction, the accuracy of light measurement is dependent upon the level of thermionic emission from the photomultiplier tube. This fixed error in measurement is a function of temperature (24) and is independent of the total cpm. This photomultiplier background emission will be a much lower percentage of the total cpm when comparing the activities of a saturated system to that of partial saturation values. The usual volumetric errors involved with the preparation of the enzyme reaction mixture will also be much less significant when the maximal activity is independent of luciferin concentration and time. Therefore, when assaying ATP via the firefly bioluminescent reaction, the maximum ease of calculation, maximum sensitivity, maximum accuracy, and reproducibility are all obtained only when the luciferase enzyme system is assayed under the conditions of luciferin saturation. ATP Assay with Crude Enzyme Preparation

It is evident from the foregoing discussions that the Sigma firefly lantern extract (FLE-50), even when reconstituted as prescribed by the manu-

110

KARL AND HOLM-HANSEN

facturer and used immediately thereafter, does not satisfy the assay assumption of luciferin saturation as described by Strehler and McElroy (14). However, the deviations from the predicted first order response will depend upon the absolute concentration of ATP as well as the concentration of luciferin and luciferase in the reaction mixture. If the ATP levels are low, relative to the concentration of luciferin (i.e., less than 500 ng of ATP per 0.5 ml of FLE-50 reconstituted to 12 ml), it is possible to generate accurate data even though the enzyme system is not saturated with respect to luciferin. If these reaction conditions remain constant, one could in principle accurately and reproducibly assay a series of ATP samples. However, the concentration of luciferin in the reaction mixture is constantly changing during the time period of the assay, primarily as a result of endogenous light emission. This necessitates standardizing the apparent amount of light emitted per ATP molecule hydrolyzed at various time periods throughout the duration of the assay. If the reaction mixture were saturated with respect to luciferin, then the Michaelis-Menten constant (K,) and the maximum enzymatic activity (V,,,) would be true constants, independent of exogenous luciferin and time. It is essential for each laboratory to evaluate the magnitude of the actual difference (if any) that results from a comparison of a series of ATP assays with and without luciferin saturation. These experiments must be carried out using the enzyme preparation procedures normally employed in that laboratory. In our laboratory we routinely assay sample extracts containing between 0.5 and 100 ng of ATP ml-‘. Within this range of ATP concentrations, the crude firefly lantern extracts can be used to accurately quantitate ATP, provided corrections are made for temporal changes in the response of the enzyme mixture to the addition of standard ATP solutions. An ATP standard curve is monitored at the beginning and at the end of each set of samples, as well as between every 40-50 assays. Corrections for each sample are then calculated and the final ATP concentration is determined using the computer data reduction method described by Booth (25). For many environmental studies, this refinement in methodology will not alter relative changes in ATP concentrations that have been observed. It will, however, extend the assay procedure to additional ecological applications (such as deep sea sediments) where the combination of low in situ ATP concentrations, high ionic interference, and adsorption onto sediment particles oftentimes results in undetectable quantities of ATP. This additional assay sensitivity is also desirable whenever metabolic studies are conducted, as well as in the calculation of the adenylate energy charge parameter. Although the current study was limited to lyophilized firefly lantern extracts from Sigma Chemical Company, a number of other commercial crude enzyme preparations are available (i.e., Worthington, FFX; Boehringer. luciferase; Calbiochem, firefly lantern extract). Lundin and Thore (6) recently compared the analytical characteristics of three of these commercial reagents. Their results indicate that the Sigma and Worthing-

EFFECTS

OF LUCIFERIN

ON ATP ASSAY

111

ton preparations are essentially identical in most respects; whereas the Boehringer luciferase has an altered pattern of sensitivity to ionic interference, a different time course of the decay of bioluminescence, and a higher background light emission (6). In addition, their data suggest that even purified luciferase preparations do not abolish all types of interference. In conclusion, there are a number of parameters that must be considered prior to the actual preparation and use of the saturated luciferin-luciferase reaction mixture. (I) Background light emission will decrease with aging time (Fig. 5) and dilution, but will increase as a result of endogenous substrate (either ATP or luciferin) in the crude enzyme preparation. (2) The concentration of inhibitory dehydroluciferin is a direct function of the cumulative background light emission and is also directly influenced by illumination and oxygen levels during the aging and assay time periods (autooxidation). Although Airth ef al. (26) have demonstrated that the inhibitory effect of dehydroluciferin can be reduced through the addition of coenzyme A, this was not attempted in the present study. Since dehydroluciferin is a competitive inhibitor of luciferase (17,19,20), this inhibition could be mitigated via the addition of a greater amount of luciferin to the reaction mixture. (3) The absolute concentration of luciferin that must be added to achieve enzyme saturation levels will depend upon the age of the extract (Table 2), the enzyme concentration (i.e., total volume of the extract), and the level of endogenous substrate (luciferin). (4) Although the actual enzyme preparation procedure will vary from laboratory to laboratory, it should be remembered that the quantitative relationship between light emission and ATP hydrolysis will yield the most sensitive, precise, and accurate information only when the firefly luciferase system is utilized during the condition of luciferin saturation. ACKNOWLEDGMENTS The authors thank Drs. F. Azam, D. A. Kiefer, and K. H. Nealson for helpful discussion and criticism during the preparation of this manuscript. This research was supported by the U.S. Energy and Research Development Administration. Contract ERDA E(I I-1)GEN 10, PA 20.

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KARL AND HOLM-HANSEN

112

5. McElroy, W. D., Seliger, H. H., and White, E. H. (1969) Photochem. Photobiol. 10, 153- 170. 6. Lundin, A., and Thore, A. (1975) Anal. Biochem. 66, 47-63. 7. Rhee, S. G., Greifner, M. I., and Chock, P. B. (1975) Anal. Biochem. 66, 259-264. 8. Allen, P. D. (1973) Develop. Indust. Microbial. 14, 67-73. 9. Holm-Hansen, O., and Booth, C. R. (1%6) Limnol. Oceanogr. 11, 510-519. 10. Chappelle, E. W., and Levin, G. V. (1%8) Biochem. Med. 2, 41-52. 11. Denburg, J. L., and McElroy, W. D. (1970) Arch. Biochem. Biophys. 141, 668-675. 12. Cheer, S., Gentile, J. H., and Hegre, C. S. (1974) Anal. Biochem. 60, 102-l 14. 13. White, E. H., McCapra, F., and Field, G. F. (1%3)5. Amer. Chem. Sot. 85, 337-343. 14. Strehler, B. L., and McElroy, W. D. (1957)in Methods in Enzymology (Colowick, S. P.. and Kaplan, N. O., eds.), Vol. III, pp. 871-873, Academic Press, New York. 15. Seliger, H. H., and McElroy, W. D. (l%O) Arch. Biochem. Biophys. 88, 136-141. 16. Suzuki, N., Suto, M., Nishikawa, K., and Goto, T. (1969) Tetrahedron Letters 53, 4683-4684.

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23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34.

Rasmussen, H., and Nielson, R. (1%8) Acta Chem. Stand. 22, 1745-1762. Aledort, L. M., Weed, R., and Troup, S. (1%6) Anal. Biochem. 17, 268-277. Schram, E. (1970) in The Current Status of Liquid Scintillation Counting (Bransome, E. D., ed.), pp. 129-133, Grune and Stratton, New York. Booth, C. R. (1975) in ATP Methodology Seminar (Borun, G. A., ed.), pp. 104-130, SAI Technology Co., San Diego, Calif. Airth, R. L., Rhodes, W. C., and McElroy, W. D. (1958) Biochem. Biophys. Acta 27, 519-532. Addanki, S., Sotos, J. F., and Rearick, P.D. (1966) Anal. Biochem. 14, 261-264. Beutler, E., and Baluda, M. C. (1964) BIood 23, 688-697. Conklin, A. R., and MacGregor, A. N. (1972) Environ. Contamin. Toxicol. 7, 296-300. Karl, D. M., and LaRock, P. A. (1975) J. Fish. Res. Bd. Cunad. 32, 599-607. Lee, C. C., Harris, R. F., Williams, J. D., Armstrong, D. E., and Syers, J. K. (1971) Soil Sci. Sot. Amer. 35, 82-86. Lin, S., and Cohen, H. P. (1968) Anal. Biochem. 24, 531-540. Lyman, G. E., and DeVincenzo, J. P. (1%7) Anal. Biochem. 21, 435-443. Rudd, J. W., and Hamilton, R. D. (1973) J. Fish. Res. Bd. Canad. 30, 1537-1546.