A COLORIMETRIC METHOD FOR THE DETERMINATION OF

DESOXYRTBONUCLEIC ACID
BY P. K. STUMPF*
(From the Department of Epidemiology and the Virus Laboratory, School of
Public Health, University of Michigan, Ann Arbor)
(Received for publication, April 28, 1947)
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 concen-

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t,ration of t.he nucleic acid. The present communication dealswith the quan-
titative application of Dische’s reaction to the specific estimation of DNA.

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) sol-
ut,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 (the-
oretical, 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
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

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FIG. 1. Relationship between the light absorption at 490 rnp and the concentration
of DNA.

I I I I I
400
450&L/&i> h7;
FIG. 2. Absorption curve of the chromogen formed by the interaction of DNA
with cysteine and 70 per cent sulfuric acid.

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
 P. K. STUMPF 369

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 cal-
culated.
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 max-
imum 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-Sum-
merson calorimeter can be employed with the 540 rnp filter, the sensitivity

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of the method falls considerably.

Factors Involved for Pmduction of Color Intensity

C2/steine-It has been observed that varying the concentration of cys-
teine changes the color intensity produced by a given concentration of
DNA. Optimum concentration of cysteine for masimum color develop-
ment 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 in-
tensity 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 con-
centrat,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 contrib-
uted by DNA was subtracted from t.he tot,al green color (at 660 mp)).
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.

TABLE I
Efect of Concentration of Cysteine on Color Intensity

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Volume of 5 per cent cysteine Optical density, log ‘$ Maximum color intensity
hydrochloride*
-
cc. per cent
0.00 0
0.01 59
0.025 0.290 93.5
0.05 0.310
0.1 0.250 80.7

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

TABLE II
Effect of Different Concentrations of Sulfuric Acid on Color Intensity

Concentration of added IO Color of solution Maximum c&r

sulfuric acid* Optical density, log i intensity
-_
per cod per cm:
50 0.00 0
60 0.129 Pink 37.5
70 0.345 “
80 0.332 I‘ 96
90 0.260 Yellow 75
100 0.177 “ 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.
 P. K. STUMPF 371

BIBLIOCXCAPHY

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

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