A COLORIMETRIC METHOD FOR THE

A COLORIMETRIC

METHOD FOR THE DESOXYRTBONUCLEIC

DETERMINATION ACID

OF

BY P. K. STUMPF* (From the Department of Epidemiology and the Virus Laboratory, Public Health, University of Michigan, Ann Arbor) (Received for publication,

April

School of

28, 1947)

EXPERIMENTAL

Principle-The method depends on the production of a pink color by the reaction of cysteine and sulfuric acid with DNA. Si;lce pentoses do not yield a color reaction with cysteine and sulfuric acid, yeast nucleic acid is unreactive. Therefore, despite the presence of ribonucleic acid (RNA), a direct estimation of desoxyribonucleic acid can be ma de. Reagents 1. 5 per cent cysteine hydrochloride

(Eastman Kodak Company) solut,ion in water. 2. 70 per cent sulfuric acid. 3. DNA standard. 0.05 per cent solution of sodium desoxyribose nucleate’ in water solution kept at 4’. The N:P ratio of the sodium salt was 1: 1.66 (theoretical, 1: 1.69). 4. RNA standard. 0.05 per cent of sodium ribose nucleate2 in water solution kept at 4”. The N: P ratio of the sodium salt was 1: 1.69 (theoretical, 1 : 1.69). Procedure

To a test-tube are added 0.05 cc. of 5 per cent cysteine hydrochloride, an aliquot of the unknown solution, the volume of which should not be more than 0.5 cc., and 5 cc. of 70 per cent sulfuric acid. The mixture is then * This work has been supported by a grant from The National Foundation for Infantile Paralysis, Inc. 1 We are indebted to Dr. Martin Hanig of this Department for a generous sample of sodium thymonucleate. * Obtained from the Schwars Laboratories, Inc., New York. 307

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Though several useful reagents have been developed to determine the presence and concentration of desoxyribonucleic acid (DNA) (l-3), all have been found to react with yeast nucleic acid and other natural products. Dische in 1944 (4) reported that, in the presence of cysteine and sulfuric acid, DXA gives a stable pink color which is proportional to the concent,ration of t.he nucleic acid. The present communication dealswith the quantitative application of Dische’s reaction to the specific estimation of DNA.

368

DETERMINATION

OF

DESOXYRIBONUCLEIC

ACJD

stirred rapidly with a glass rod, and, after standing 10 minutes at room temperature, optical densities are measured at the 490 rnF wave band with

between the light absorption

I 400

I

I

I

450&L/&i> FIG. 2. Absorption curve of the chromogen with cysteine and 70 per cent sulfuric acid.

at 490 rnp and the concentration

I h7;

formed by the interaction

of DNA

the Beckman quartz spectrophotometer. A reagent blank is used to set the instrument scale to 0. The concentration of DXA can then be determined by interpolation from


FIG. 1. Relationship of DNA.

P.

K.

369

STUMPF

Factors

Involved for Pmduction

of Color Intensity

C2/steine-It has been observed that varying the concentration of cysteine changes the color intensity produced by a given concentration of DNA. Optimum concentration of cysteine for masimum color development has been found to be 0.05 cc. of 5 per cent cysteine hydrochloride. As is inclicat’ed in Table I, there is a sharp fall if this concentration is not employed. Concentration of Sulfuric Aci&--4s is shown in Table II, the color intensity is ser?sitivc to a change in the concentration of sulfuric acid. No color is formed if the acidity is too low, while concentrated sulfuric acid yields a yellow color wit,h 11X-4. Maximum color intensity is developed when 5 cc. of 70 per cent sulfuric acid are used. The final or critical concentrat,ion of acid is, therefore, 63 per cent. Concentrated hydrochloric acid produces no color \\ith the cysteine-DXA system. Temperature-The det,ermination is’ carried out at room temperature, which averages between 23-25’. There is little, if any, increase in color if the reaction mixture is incubated for 5 minutes at either 3i”, 45’, or 65’. Stability in Color--The color is unusually stable and does not fade or change. There is, however, a slight increase in color intensity with time. Therefore, it is important to carry out all readings after a standard time interval in order to obt.ain reproducible results. Specijkity of Method-The method is of considerable value with mixtures of RXA and DKA, since a direct, analysis of DNA without interference from RNA can be made. Ry employing this method together with Bial’s reaction (5) for RNA, accurate determinations of both acids can easily be carried out. For example, in an analysis of a known mixture of 150 y of DNA and 100 y of RNA, 143 y of DNA were found directly by this method and 103 y of RT\‘A by Bial’s reaction (after the color intensity contributed by DNA was subtracted from t.he tot,al green color (at 660 mp)).


a standard curve prepared by plotting the readings against the known concentrations of DXA as is shown in Fig. 1. However, a standard of about 250 y of DNA may also be prepared and, from the readings of the standard and the unknowns, the concentration of the latter may be calculated. As is indicated in Fig. I, the curve follows Beer’s law from a concentration of 25 to 550 y. Below 25 y the curve flattens out and above 550 y the color becomes too intense for accurate readings. Fig. 2 shows that maximum absorption occurs at 490 rnp, and that above and below this sharp peak t,here is a rapid drop in light absorption. Although the Klett-Summerson calorimeter can be employed with the 540 rnp filter, the sensitivity of the method falls considerably.

370

DETERMINATION

OF

DESOXYRIBONUCLEIC

ACID

Not only is the reagent specific for DNA, but it fails to react with 1 mg. of the following compounds: phosphoglyceric acid, glycerophosphate, glucose-l-phosphate, glucose-6-phosphate, glucose, arabinose, alanine, xanthine, nicotinic acid, coenzyme 1, adenosine triphosphate, and creatine. Fructose and its derivative, fructose-l, 6-diphosphate, gave a slight yellow color. However, in any nucleic acid estimation of tissues, the nucleic acid extraction procedure developed by Schneider (6) should be employed to remove interfering substances, such as fructose derivatives. I

TABLE

Volume

of Concentration

of 5 per cent cysteine hydrochloride*

of Cysteine Optical

density,

on Color

log ‘$

Intensity Maximum

color

intensity

-

cc.

per cent

0.00

0.01 0.025 0.05 0.1

0 59 93.5

0.290 0.310 0.250

80.7

* 250 y of sodium DNA, 5 cc. of 70 per cent sulfuric acid, and the indicated of 5 per cent cysteine hydrochloride. II of Sulfuric

amounts

TABLE Effect Concentration sulfuric

of Different

of added acid*

Concentrations Optical

density,

IO log i

Color

Acid

on Color

of solution

-_

per cod

50 60 70 80 90 100

Intensity Maximum c&r intensity

per cm: 0.00

0

0.129 0.345 0.332 0.260 0.177

Pink “ I‘ Yellow “

37.5

-

96 75 52

* 0.05 cc. of 5 per cent cysteine hydrochloride, 250 7 of sodium DNA, and varying concentrations of sulfuric acid to make a total volume of 5.55 cc.

The author is deeply indebted to Dr. Z. Dische for calling his attention to the fundamental observation of the cysteine-sulfuric acid-DNA reaction. SUMMARY

A specific calorimetric method for the determination of desoxyribonucleic acid has been described. Since ribonucleic acid yields no color with the reagent, desoxyribonucleic acid may be estimated, wit,hout interference, in the presence of ribonucleic acid.


Efect

P.

K.

STUMPF

371

BIBLIOCXCAPHY

1. 2. 3. 4. 5. 6.

Gurin, S., and Hood, D. B., J. Biol. Chem., 139, 775 (1941); 131, 211 (1939). Cohen, S. S., J. Biol. Chem., 166, 691 (1944). Dische, Z., Mikrochemie, 8, 4 (1930). Dische, Z., Proc. Sot. Ezp. Biol. and Med., 66, 217 (1944). Mejbaum, W., 2. physio2. Chem., 268, 117 (1939). Schneider, W. C., J. Biol. Chem., 181,293 (1945).


A COLORIMETRIC METHOD FOR THE DETERMINATION OF DESOXYRIBONUCLEIC ACID P. K. Stumpf J. Biol. Chem. 1947, 169:367-371.

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