An ultrasensitive radioassay for hexokinase

ANALYTICAL

52, 272-279 (1973)

BIOCHEMISTRY

An

Ultrasensitive RONALD

Department

Radioassay

E. GOTSl

of PhaTmacoZogy,

AND

University Los

for

SAMUEL of Southern

Angeles, California

Hexokinase

P. BESSMAN

C.alijornia School 900%

oj Medicine,

Received June 2, 1972; accepted October 6, 1972 A batch chromatographic method for the determination of hexokinase employing trace-labelled glucose and resin chromatography is described. Markedly enhanced sensitivity compared with the standard spectrophotometric assay is shown. Its greater reliability and use for particulate-tissue preparations is discussed.

This report describes an improved radioassay for hexokinase (2.7.1.2 ATP: glucose-6-phosphotransferase) based on the formation of tritiated glucose-6-phosphate from tritium-labeled glucose, isolation, and counting of the captured product. Increased sensitivity and usefulness for particlebound enzyme endow this assay with significant advantages over previously described methods. METHODS

1. Radioassay Procedure. The final reaction mixture consists of Trischloride buffer 0.107M pH 7.50, magnesium chloride 0.0322 M, adenosine triphosphate 5.36 mM, and glucose 1.6 mM. To prepare the system, the glucose is combined with glucose, tritiated in the one position, so that the specific activity of the substrate varies depending on the desired sensitivity (generally 1 to 6 ,&i/~mole) ; 0.075 ml of this substrate mixture is added to 0.50 ml of the other components. The reaction is initiated by the addition of 0.10 ml of the enzyme and is incubated at room temperature for ten minutes. The reaction is terminated by the addition of 4 ml of a 1 M glucose solution in 0.17M ammonium hydroxide. The base is required to ionize the glucose-6-phosphate. For all assays zero time blanks are run. 0.25 g of untreated dry Dowex 2X8-100, 50-100 mesh is added directly to the reaction mixtures and the systems are vigorously agitated three times in a five-minute period to insure complete adsorption. After the final mix, the resin quickly sediments and the supernatant is aspirated and discarded. The Dowex resin is thoroughly washed four ‘Recipient of a National Institutes Fellowship number 1 F02 GM48602-01

of Health Special Postdoctoral Training which, in part, supported this work. 272 Copyright @ 1973 by Academic Press, Inc. ,411.GnL+o ,.c . ..%.,A..“+:,.. :.. n...- *,...m mo,..rr,A

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times with 4 ml of a 0.17 M solution of ammonium hydroxide. It is then treated with 1 ml of a 1 M hydrochloric acid solution by vigorously agitating every two minutes for eight minutes. A 0.50-ml aliquot of the acidic eluant containing the eluted glucose-6-phosphate is added to 10 ml of a standard scintillation fluid composed of 100 g naphthalene, 0.3 g dimethyl POPOP, 5.0 g PPO, 730 ml dioxane, 135 ml toluene, and 35 ml absolute methanol. The sample is counted in a liquid scintillation counter. For each assay series, blanks are run which contain no enzyme. The blank counts are subtracted from the counts of the test samples. Quenching is determined by the channels ratio method, but once established, uniformity of preparation permits a standard efficiency figure to be used. Control assays incubated without ATP give no reaction. To determine the number of micromoles of glucose converted per minute, the following formula is applied: micromoles formed minute (cpm sample - cpm blank) X dilution factor = efficiency X specific activity of substrate X assay time where cpm is counts per minute, the dilution factor is 2-0.5 ml counted from a total elution volume of 1 ml-and the specific activity is the distintegrations per minute per micromole of substrate glucose. An enzyme unit is defined as the amount of enzyme that catalyzes the conversion of one micromole of glucose per minute. 2. Optimization of the Assay. To opt,imize the assay conditions a variety of parameters were studied. Those included variations in volumes of adsorbant and eluant, variations in mixing times of both adsorption and elution phases, variations in the concentration of the HCl eluant, and changes in the weight of resin utilized. 3. Determination of Assay Reliability. The accuracy, reliability, and usefulness of the assay were tested in three ways. First, kinetic studies were performed on crystalline yeast hexokinase and the experimental K, was compared to the published value. Second, the standard spectrophotometric assay was compared to the radioassay using crystalline yeast enzyme. Finally, the assays were compared using particulate (mitochonclrial bound) rat-muscle enzyme. The rat-muscle mitochondria were prepared by Waring Blender homogenization for 30 set in 5 ml/g weight of Tris-chloride 0.01 M pH 7.4, mannitol 0.35 M and EDTA 10h4M. The crude extract was spun twice at 5OOg for 10 min and the supernatant was spun at 5000g for 10 min to recover the mitochondrial pellet. This was washed three times in the original buffer and then suspended in 0.01 M Tris-Cl pH 7.4 containing

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0.5% tween 80 to give a protein concentration of 1 mg,/ml. The pellet was vigorously agitated with a vortex mixer and all subsequent dilutions were made in this suspending buffer. 4. Spectrophotometric Assay. The method utilized was a standard coupled assay in which the glucose-6-phosphate produced by the hexokinase reaction serves as substrate for a second reaction involving glucose-6-phosphate dehydrogenase. NADPH is formed in the process and is followed spectrophotometrically at 340 nm. The hexokinase is rate limiting and the number of micromoles of NADPH formed per minute is identical to the number of glucose molecules converted to glucose-Bphosphate. Precise quantitation is achieved by employing the extinction coefficient of NADPH, the reaction volume and the change in optical density according to the following formula: micromoles formed = Change in OD per minute minute 6.22

X 1.20

The assay mixture is identical to the one used for the radioassay with the addition of 1.34 mM NADP and 0.3 units of glucose-8phosphate dehydrogenase. The react,ion is started by the addition of enzyme and is followed from the second to the fifth minute after initiation. Controls were run with no ATP, no glucose, no glucose-6-phosphate dehydrogenase, or no NADP. These all failed to register any activity. An enzyme unit was expressed as before-the amount of enzyme which catalyzed the conversion of one micromole of glucose per minute. MATERIALS

1. Reagents. The hexokinase was crystalline yeast enzyme supplied by Calbiochem. The ATP, glucose, glucose-6-phosphate dehydrogenase, NADP and Dowex were purchased from the Sigma Chemical Company. The tritiated glucose was obtained from the Nuclear Chicago Corporation. S. Equipment. Spectrophotometric determinations were made on the Gilford 210 recording spectrophotometer. Radioassays were determined on the Nuclear Chicago Unilux II liquid scintillation counter and statistical analyses were performed on the Olivetti Underwood Programma 101 computer. Centrifugation for mitochondrial preparation was carried out in an International refrigerated centrifuge PR-6 with a high-speed attachment. RESULTS

1. Optimization of Assay Parameters. In each optimization

the enzyme concentration

was constant

procedure, and only the parameter under

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investigation was varied. The conditions studied were volumes of adsorbent and eluant, adsorption time, elution time, concentration of eluting HCl and weight of the Dowex resin. The results are expressed as percent of maximum activity in each series and are shown in Table 1. The volume of eluant and of adsorbent (above 1 ml) made little statist’ical difference. For subsequent assays 4 ml was chosen as the adsorption volume and 1 ml the elution volume. A ELmin adsorption time was optimal and an elution time of 8 min was statistically similar to the 30min elution. Subsequently, five and eight minutes were used for these processes, respectively. The resin weight and HCl molarity were of mini-

Optimization Adsorption

Elution

time (min) 0 5 10 time (min) 0

Percent

30 volume

(ml) 100 86 91 91 79

2

a

Adsorption

Eluant

activity I 173 100 93 32 67 81 88 95 100

l 6 8 Elution

TABLE 1 of Assay Parameters

5 volume 0.5

2 s 4 concentration

(ml) 66 73 92 85 100 (N HCl)

0.1

x7 90

0.5

100

Dowex

2 resin weight 0.25 0.50 1.00 1 50

91

(g) 98 100 95 91

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ma1 significance. A one-normal solution of the eluant and 6.25 g of Dowex were employed. 2. Kinetic Studies. As one measure of the efficacy of this assay method a series of initial velocities were measured using substrate concentration as the independent variable. These results were tabulated in a double reciprocal form and were subjected to regression analysis. The linear regression plot with the standard deviations (three determinations for each point) is shown in Fig. 1. Calculation of the Michaelis Menton constant proved to be 1.74 X lo-” M for glucose. This is in excellent agreement with the published figures for this enzyme which vary from 1 to 4 X 10m4 M the most commonly reported value being 1.60 X 10m4 M. 3. Comparison of the Radioassay and Spectrophotometric Assay. Determinations were made at varying enzyme concentrations by both the radioassay and spectrophotometric methods. The results are presented in Figs. 2 and 3 for the crystalline enzyme and 4 for the mitochondrial enzyme. Above the 1-milliunit level the two assays agree favorably. Below this level the spectrophotometric assay begins to lose linearity and becomes divergent, whereas the radioassay is readily extended to the lmicrounit level. It is also evident by the point scat’tering in Fig. 4 that particulate-bound enzyme is determined imprecisely by the spectrophotometric method below the 1 milliunit level. The isotopic assay is, therefore, useful in a thousandfold more sensitive range than the earlier method. Increasing the counting t,ime and specific activity of the substrate could, theoretically, enhance the sensitivity another hundredfold. This nanounit sensitivity was not required for our purposes, but its availability presents the potential of enzyme determination in single cells.

FIQ. 1. A double reciprocal plot of initial utilizing the radioassay for hexokinase.

velocities versus substrate concentratioqs

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NANOGRAMS

FIG. 2. Comparison yeast hexokinase.

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HEXOKINASE

of the spectrophotometric

and radioassays using crystalline

DISCUSSION

The enzymatic conversion of glucose to glucose-&phosphate has been followed in many ways including manometric determination of the COZ liberated from an HCOs- containing reaction system; continuous titration of liberated H+; chemical glucose determinations; measurement of ATP or ADP (l-6). In recent years the most popular method has been the coupled spectrophotometric technique described in this paper (7,8). In

25

.Ol NANOGRAMS

.50

HEXOKINASE

FIG. 3. Radioassay of crystalline yeast hexokinase those attainable by other methods.

at levels of sensitivity

below

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I

10-4

10.' MILIORA~~3~.0T61NIo-z

Fro. 4. Comparison of the spectrophotometric mitochondrial-bound hexokinase.

and radioassays csing rat-muscle

1957, Lowry et al. described an ultrasensitive analytical procedure in which NADP, NADPH, NAD, or NADH could be determined spectrofluorometrically (9). This would theoretically allow nanounit determinations of hexokinase but the authors did not adapt their method to use with this enzyme. Their procedure, moreover, is far more demanding than the present method which can achieve similar sensitivity. In 1963, Sherman first suggested the possibility of using an anion exchange method to determine radioactive glucose-6-phosphate (10). This concept was amplified by Newsholme et al. who used DEAE cellulose disks to capture the product (11). Their method was delicate and tedious and was not generally adopted. Moreover, the sensitivity of their assay, although more sensitive than the coupled spectrophotometric method, did not have close to the potential of the method described here in which a far larger fraction of the total yield is counted. In addition to added sensitivity, there are several other major advantages of this assay over the commonly employed coupled assay. Coupling provides additional sources of side reaction interference. For instance, if one measures liver hexokinase by the spectrophotometric method, one often sees a linear disappearance, instead of synthesis of NADPH. This undoubtedly occurs because the concomitant operation of an NADPH utilizing reductive reaction. The direct radioassay obviates some of these interfering problems. Secondly, in our laboratory, we have been determining mitochondrial-bound enzyme. Despite vigorous solubilization techniques, the presence of particulate contaminants often interferes with the spectrophotometric readings. This may, in part, account for the scattering in Fig.

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4. No such difhculties arise with the radioassay, which is ideally suited to the determination of particulate-enzyme preparations. Finally, mention should be made of the universal adaptability of this unusual batch chromatographic assay technique to any situation in which product and reactant differ appreciably in electrostatic configuration. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.

CRANE, R. K., AND SOLS, A. (1953) J. Biol. Chem. 203, 273. SLATER, E. C. (1953) Biochem. J. 53, 521. RACKER, E. (1947). J. Biol. Chem. 167, 843. COLOWICK, S. P., AND KALCICAR, H M. (1943) J. Biol. Chem. 148, 127. KUNITZ, M., AND MCDONALD, M. R. (1946) J. Gen. Physiol. 29, 393. CRANE, R. K., AND SOLS, A. (1955) Methods in Enz2/mol. 1, 277. SLEIN, M. W., CORI, G. T., AND CORI, C. F. (1950) J. Biol. Chem. 186, 763. JOSHI, M. D., AND JAGANNATHAN, V. (1960) Methods in Enzymol. 1X, 371. LOWRY, 0. H., ROBERTS, N. R., AND KAPPHAHN, J. I. (1957) J. Biol. Chem. 224, 1047. 10. SHERMAN, J. R. (1963) Anal. Biochem. 5, 548. 11. NEWSROLME, E. A., ROBINSON, J., AND TAYLOR, K. (1967) Biochem. Biophys. Acta 132, 338.