Rapid colorimetric determination of adenine compounds

AN.PLYTICAL

BIOCHEMISTHY

Rapid

5, 64-69

(1963)

Calorimetric

Determination

of

Adenine

Compounds1 JOSEPH Fwtn

R. DAVIS

AND

ROBERT

N. MORRIS2

fhe Depurtment of Pharmacology and Therapeutics, Stritch School of Medicine, and Graduate School, Loyola University, Chicago, Illinois Received

May

28, 1962

Numerous spectrophotometric methods employing the ultraviolet range have been described for the determination of adenine. Since the absorption maximum of all the nucleic acid bases varies between 250 and 290 mp, one disadvantage to this method is the lack of specificity for adenine in a mixture of nucleic acid bases. In addition, many other biological materials can absorb at similar wavelengths and thus interfere with the determination of adenine. Differential spectrophotometry has been reported by Kalckar (1) to differentiate adequately a number of purine compounds by changes in the ultraviolet absorption of t,hese compounds resulting from actions of specific enzymes. A specific calorimetric analysis for adenine has been reported by Woodhouse (2) employing reduction with zinc dust and coupling with LV- (1-naphthyl) ethylenediamine. Although these methods have been found to give satisfactory determinations of adenine, they are both complicated and time consuming. For these reasons, studies have been carried out in this laboratory on a rapid and specific calorimetric analysis of adenine which would serve not only as a quantitative assay but also as a convenient spot test for this substance. In the course of determining citrate content in tissue slices by the pentabromacetone ether extraction method of Natelson et al. (3), it was observed that a clear yellow color appeared after the addition of hydrogen peroxide when ATP had been added to the incubation medium. The possibility that this color reaction was due to some color complex involving adenine was confirmed by using the same procedure with adenine alone. The present experiments were designed to ascertain the 1 These studies were Cancer Society (IN-52) Health Service. ‘Predoctoral Trainee,

supported in part by Institutional Grants from and the General Research Support Grant United

States

Public 64

Health

Service,

Training

the American of the Public Grant

2G-77.

COLORIMETRIC

ANALYSIS

OF

ADENINE

65

specificity of the color reaction for adenine and the sensitivity of the analysis, as well as to investigate the optimal conditions of the reaction. METHODS

Apparatus. A Beckman DU spectrophotometer was used for absorbance measurements. Purines and Pyrimidines. Adenine, guanine, xanthine, hypoxanthine, cytosine, 5-methylcytosine, uracil, and thymine were obtained from the California Corporation for Biochemical Research (Los Angeles, Calif.). Purine, uric acid, caffeine, theobromine, theophylline, 6-chloropurine, adenosine, adenosine monophosphate, adenosine diphosphate, and adenosine triphosphate were obtained from Mann Research Laboratories (New York, N. Y.). 6-Mercaptopurine was obtained from Nutritional Biochemicals Corporation (Cleveland, Ohio). In all instances, 0.11v H,SO, was employed as the solvent. Reagents. Stock solutions were employed for 18 1V H,SO,, 0.2 M KBr, 1 N KMnO,, and 67%H,O,. The 1 N solution of potassium permanganate was obtained from the Fisher Scientific Co. (Chicago, Ill.) and contained 31.61 mg KMnO,/ml by sodium oxalate reference standard. Procedure. A l&ml aliquot of the sample to be analyzed was transferred to a centrifuge tube with a conical bottom and 0.2 ml of 18N H,SO, was added. The contents were mixed and 0.1 ml of 0.2 fV KBr was added. Following shaking, 0.3 ml of 1 N KMnO, was added and after waiting 5 min 6% H?O, was introduced dropwise to decolorize the excess permanganate. The final volume was then adjusted to 3.0 ml with distilled water and the optical density was measured within 15 minutes at 330 rnp against a reagent blank. RESULTS

AND

DISCUSSION

A yellow color appeared almost immediately following the decolorization of the excess permanganate with hydrogen peroxide in the reaction mixture containing adenine. The absorption of light by adenine before and after the outlined procedure is shown in Fig. 1. A solution of untreated adenine in 0.1 N H,SO, had a single absorption maximum of 262 mp. Following color development, the adenine solution had maximal absorption at wavelengths of 254, 330, and 420 mp. The absorption peak at 330 rnp was chosen as the optimal wavelength for sample readings, although the yellow color resulting from the absorption at 420 rnp was sufficient to carry out a rapid qualitative test for the presence of adenine. In order to determine the reproducibility of the method, ten samples of adenine (two different commercial lots) were treated with the above reagents and the optical density at 330 rnp determined. The average

DAVIS

AND

100. 1 1 _ p8.‘“ji ( ::: ;; \\

I

j

220

I

3 300

I

I

.........ADENlNE ADENINE -CHROMOPHORE

:

260

MORRIS

1 340

I

I

360

420

_

1 460

500

w FIG. 1. Absorption of light by adenine before and after treatment the outlined procedure. In the regions of high absorption, measurements on appropriate dilutions of a solution which was originally 5 x 10e3

according to were made M adenine.

molar extinction coefficient was found to be 2170 (standard error of +20) at a concentration of 5 X 1O-4M adenine. Because of the nonstoichiometric nature of the reaction, the molar extinction coefficient varied somewhat with adenine concentration. Figure 2 is a standard curve of optical density versus adenine concentration. A straight line was obtained from 10 to 50 pg of adenine/ml of reaction mixture. The stability of the adenine chromophore was investigated with regard to its absorption at 330 mp. Essentially no change in optical density occurred up to 60 min following the final addition of hydrogen peroxide. Figure 3 indicates the effect of varying amounts of potassium bromide on the yield of the adenine chromophore. An inverse relationship occurred between the absorption at 262 m,u as compared to the absorption at 330 mp, which was dependent upon the concentration of KBr in the reaction mixture. Similar relationships were also found to be dependent upon the concentration of H,SO, and KMnO, in the reaction mixture. These data indicate that the absorption peak at 330 rnp is due to the

COLORIMETRIC

MICROGRAMS

FIG. 2. Standard mixture

against

the

2 FIG. 3. Effect 5 x

lo-‘44

ADENINE/ml

OF

REACTION

of varying adenine.

amounts

67

ADENINE

MIXTURE

curve for adenine chromophore plotted optical density measured at 330 m@.

MICROMOLES from

ANALYSIS

in pg adenine/ml

reaction

4 6810 20 40 6080 KBr/ml REACTION MIXTURE of potassium

bromide

on yield

of chromophore

formation of the adenine chromophore. It would appear, however, that the absorption at 262 rnp may represent not only unreacted adenine but a portion of the adenine chromophore as well. The specificity of the reaction for adenine was investigated by testing a number of purines and pyrimidines (Table 1). All of the compounds

68

DAVIS

AND

MORRIS

studied were found to give a negative test with respect to both the visible yellow color and an ultraviolet absorption peak at 330 mp. TABLE 1 NONINTERFERING COMPOUNDP Guanine Hypoxanthine Xanthine Purine Uric acid Caffeine Theobromine Theophylline

6-Chloropurine 6-Mercaptopurine Uracil Thymine Cytosine 5-Methylcytosine

0 Each of the compounds listed wae mixed at 5 X 10m44f with adenine and the absorbancy after color development was compared with that for an adenine control.

In contrast to the results obtained with purines and pyrimidines other than adenine, it was found that such adenine-containing compounds as adenosine, adenylic acid, adenosine diphosphate, and adenosine triphosphate all produced a visible yellow color and the characteristic ultraviolet absorption peak at 330 rnp when treated with the outlined procedure. The molar extinction coefficients at 330 rn@ for these compounds were 102, 97, 91, and 94%, respectively, of the corresponding molar extinction coefficient obtained for adenine itself. The fact that these adenine nucleosides and nucleot.ides reacted positively without prior hydrolysis, such as refluxing for 1 hr with 1 N H&JO,, eliminates this time-consuming step when a rapid quantitative or qualitative analysis is desired for these substances. In order to test the applicability of the present method to biological fluids, recoveries were made of known quantities of adenine added to protein-free filtrates of rat serum, kidney, and spleen. Tungstic acid was employed as the precipitating agent. The recovery of added adenine ranged between 98 and 102% in these tissues. The total tungstic acidsoluble adenine of the kidney was found to be 171 pg/gm wet weight tissue with a standard error of -+12, while the total tungstic acidsoluble adenine of the spleen was found to be 179 pg/gm wet weight tissue with a standard error of k14. No demonstrable adenine was found in samples of rat serum employing a 1: 10 dilution of the original material. SUMMARY

A procedure for both a rapid qualitative and a quantitative determination for adenine in a mixture of various purines and pyrimidines is des,cribed. In addition, the method can also be applied for the rapid

COLORIMETRIC

ANALYSIS

OF

69

ADENINE

detection and quantitative estimation of a number of adenine nucleosides and nucleotides without prior hydrolysis of these compounds. ACKNOWLEDGMENTS The authors wish to express their appreciation to Dr. Norton C. Melchoir and Dr. Frederick W. Pairent of the Department of Biochemistry of Stritch School of Medicine, Loyola University, for their many helpful suggestions. REFERENCES 1. KALCKAR, 2. WOODHOUSE,

3.

NATELSON,

M., J. Biol. Chem. 167, 445 (1947). D. L., Arch. Biochem. 25, 347 (1950). S., LUCOVOY, J. K., AND PINCUS, J. B., J. Biol.

H.

Chem.

170,

597 (1947).