Determination of nucleic acids with concentrated H2SO4

ANALYTICAL

BIOCHEMISTBY

Determination

13, 314-321 (1965)

of Nucleic Acids with Concentrated I. Deoxyribonucleic

H2S04

Acid1

PAULBYVOET From. the Department of Pharmacology University of Florida College of Medicine,

and Therapeutics, Gainesuille, Florida

Received May 3, 1965

In 1962, Staron et al (1) described a quantitative method for the determination of deoxyribonucleic acid (DNA) with concentrated sulfuric acid. According to their procedure, 1.5 ml of concentrated H2S04 is added to 100 pl of an extract of DNA obtained by heating a DNA extract in 59’0 trichloroacetic acid (TCA) at 90°C for 30 min. After 30 min at room temperature, the deoxyribose in the extract is converted into a yellow colored compound which is measured at 480 mp. Under these conditions only fructose, rallinose, inositol, and tryptophan produced an absorbance at this wavelength, which however is small if compared with the absorbance produced by equal concentrations of deoxyribose. Dische, who described this reaction in 1955 (2)) pointed out that arabinal produces a yellow color too. DNA preparations obtained by phenol extraction procedures or by the Schmidt-Thannhauser method (3) are in general minimally contaminated with other sugars than deoxyribose. In view of its simplicity, as well as its sensitivity, the H,SO, reaction was studied, and found to be a satisfactory method. A few modifications, however, were introduced to increase its sensitivity and accuracy. EXPERIMENTAL

Apparatus: The Beckman Model DU ultraviolet spectrophotometer, the Coleman Junior spectrophotometer, and the Cary recording ultraviolet spectrophotometer, model 15, were used in these studies. Materials: 2’-Deoxy-n-ribose, A grade, Calbiochem; n-ribose, A grade, Calbiochem; sperm deoxyribonucleate, Nutritional Biochemicals; yeast ribonucleic acid (RNA), Schwartz BioResearch. All other chemicals were of analytical grade. Procedure: In general the reaction was carried out by adding 1.5 or 3 ml concentrated H,SO, (specific gravity 1.84) to a loo-p1 sample in ‘This

research was supported

by NIH

Grants CA 18402 and CY 6500. 314

DETERMINATION

5% TCA and reading temperature.

OF

the absorption

DNA

WITH

315

H2S04

at 470 m(r. after 30 min at room

RESULTS

Spectra of Ribose and Deoxyribose in Concentrated H,SO,: To 100 ~1 of a 5% TCA solution, containing either 100 pg deoxyribose or 200 pg ribose, was added 3 ml concentrated H&SO,. The spectra of these solutions shown in Fig. 1 indicate that the absorption maximum of .7oc

.500 E s Q B * 2 ,300

A

,100

--SW--------w-1

4 0

450

470

500mp

Wavelength in

FIG. 1. Absorption spectra concentrated H,SO,.

of deoxyribose

(solid

line)

and

ribose

(broken

line)

deoxyribose under these conditions occurs at 470 rnp rather than at 480 rnp. It appears that ribose will not interfere with the determination of deoxyribose at this wavelength. In order to establish the effect of various conditions prior to the addition of sulfuric acid, the spectra of deoxyribose in concentrated sulfuric acid were studied after dissolving the deoxyribose in 5% TCA, 1 N HCI, or 0.1 N NaOH. The alkaline solution was heated at 60°C for 30 min. The spectra recorded in Fig. 2 show that heating in alkali diminishes the maximum at 470 rnp, with the development of a peak with a maximum at 290 mp. Although DNA develops a bright yellow color if treated with concentrated sulfuric acid, deoxyribose produces a brownish yellow color under the same conditions.

316

PAUL

BYVOE!I!

\ \ \ \

\B

250

360

460

47b

560

2. Absorption spectra of 400 pg deoxyribose in 3 ml concentrated H&O,. Before addition of HzSO, the deoxyribose was dkaolved in 1 N HCI (A), in 0.1 N NaOH and heated at 60°C for 60 min (B), in water (0, and in 6% TCA (D). FIQ.

This is probably due to the absorbance in the area between 340 and 420 rnp, which is always slight in the case of DNA. As can be seen, these maxima disappear if the deoxyribose is pretreated with 1 N HCl, but become more pronounced if the deoxyribose is pretreated with TCA. Accordingly, a bright yellow color is produced after pretreatment with HCl, whereas TCA tends to cause a brown tinge. It is conceivable that the maxima at 365 and 385 rnp represent deoxyribose derivatives. Heating in 1 N HCl destroys deoxyribose rapidly, with the development of a chromophore with x,. = 261 m,u (4). Spectra of DNA and RNA in Concentrated H,SOa: DNA and RNA (3 mg/ml) were hydrolyzed in 5% TCA at 90°C for 30 min. To 100 ~1

DETERMINATION

OF

DNA

WITH

317

H2S04

of these hydrolyzates was added 3 ml concentrated H&O,. The spectra shown in Fig. 3 indicate that the absorption maximum of the DNA is also situated at 470 rnp, and that no interference is caused by RNA at this wavelength. The concentration of DNA in solutions containing .500 -

---______-------ma_* I I 450 400 Wavelength

I 470

J 500

mp

FICJ.3. Absorption spectra of DNA (solid line) and RNA (broken line) in concentrated H,SO,. Both samples were treated with 5% TCA at 90°C for 30 min.

DNA as well as RNA can therefore also be determined accurately. Lineatity of the Reaction: Increasing volumes of solutions containing untreated DNA and DNA heated in 5% TCA were brought to a final volume of 100 ,uI with a final concentration of 5% TCA. After the addition of 1.5 ml of concentrated H&SO,, the resulting absorptions were plotted against the amounts of DNA in the samples, as shown in Fig. 4. It appears that the relation between the absorbances and the concentrations is not linear, but slightly curved. As can be seen, preheating of the DNA in 5% TCA hardly affected the yield of chromogen. Development of Color with Time: Staron et al. reported that maximal color is obtained after 20-30 min at room temperature, as is shown in Fig. 5. Deoxyribose, untreated DNA or DNA heated in 5% TCA behaved in a similar fashion. The heat which is produced when the concentrated H&SO, is added to the sample probably accelerates the development of color. Indeed, as is shown in Fig. 4, the development of the maximal absorbance was delayed considerably if the tube containing the hydrolyzed DNA sample was cooled in ice during the addition of the concentrated H&JO,.

318

PAUL

BYVOE’I!

.8OC

.6OC

I

I

I

100

I

I

200

Y DNA

FIG. 4. Absorbances at 470 ma of various concentrations of DNA treated with 5% TCA at 90°C for 30 min (broken line) and untreated DNA (solid line) 30 min after addition of 1.5 ml concentrated HzS04 to 1OOyl samples.

EJgect of Total Volume of the Sample:

recorded in Table

As can be seen from the data 1, the absorbance at 470 rnp is decreased sharply by

TABLE 1 EFFECT OF SAMPLE VOLUME ON OPTICAL DENSITY (Volume of HzS04 added = 3 ml). DNA solution (concentration 1 mg/ml), d

100 100 100 4 Averages of duplicate

Volume water

of distilled added, pl

Em

0 100

0.7500

200

0.185

0.395

determinations.

addition of small amounts of distilled water. The total volume of the sample should therefore always be kept constant and should not exceed 100 jL1.

DETERMINATION

OF

DNA

WITH

319

H2S04

Minutes

FIG. 5. Development of absorbance at 470 mp with time after addition of concentrated H&O, to a sample containing DNA heated in 5% TCA. Untreated DNA as well as deoxyribose yielded the same curve. When the sample was cooled in ice during addition of concentrated H&O, the curve with the open circles was obtained.

Effect of TCA Concentration of the Sample: 50 ,uI of TCA solutions containing increasing concentrations of TCA were added to 50 ,J.J of a solution containing either 1 mg/ml DNA or 1 mg/ml deoxyribose; 1.5 ml concentrated H&30, was added and the absorptions measured at 470 rnp. The results recorded in Table 2 indicate that a slight increase in TABLE

EFFECT OF TCA (Volume

of HzSOd

khlpk

50 ~1 DNA

soln.

50 ~1 deoxyribose

0 Average

in 5%

soln.

of triplicate

2

CONCENTRATION ON OPTICAL DENSITY added

= 1.5 ml) Added

TCA

in H20

(1 mg/ml)

(1 mg/ml)

50 ~1 distilled water 50 /J 10% TCA 50 ~120% TCA 50 /.d 40% TCA 50 ~1 10% TCA 50 ~120% TCA 50 J 40% TCA

0.174a 0.176

0.242 0.272 0.400 0.455 0.482

determinations.

absorption results from the increase of the TCA concentration. Comparison with the Diphenylamine Reaction: DNA samples were prepared from the livers of 12 different rats by blending the tissues in

320

PAUL

BYVOET

isotonic saline and subsequently carrying the sediments through the routine extraction procedures with acid and organic solvents (5). The material was then subjected to the Schmidt-Thannhauser procedure (3)) precipitated, and extracted with 5% TCA at 90%. All samples were assayed for deoxyribose with the diphenylamine reaction (2) as well as the HzS04 reaction. The results recorded in Table 3 show that the TABLE COMPARISON Rat liver Sample

1 2 3 4 5 6 7 8 9 10 11 12

DNA No.

OF

HzSOc

H&O4 E470

0.465” 0.460 0.500 0.580 0.465 0.565 0.435 0.395 0.580 0.445 0.428 0.550

0 Averages of duplicate

REACTION

AND

3 THE

DIPHENYLAMINE

Reaction

Diphenylsmine

REACTION reaction

pg DNA

ES86

EI#L--I60

erg DNA

87 86 95 115 87 111 80 71 115 82 78 108

0.228” 0.212 0.246 0.295 0.214 0.264 0.200 0.177 0.277 0.208 0.205 0.206

0.109” 0.100 0.118 0.141 0.098 0.127 0.093 0.083 0.137 0.096 0.094 0.121

96 88 104 125 86 112 81 73 120 a5 83 112

determinations.

absorbancy values observed in the H2S0, reaction are approximately twice those obtained by the diphenylamine reaction. It appears that the values obtained by the two methods agree reasonably well. The values for the H,SO, reaction were calculated from a calibration curve similar to the one shown in Fig. 3. DISCUSSION

Although the reaction with concentrated HzSO, appeared to be only about twice as sensitive as the diphenylamine react.ion in our hands, its simplicity is probably unequaled by other methods designed for the determination of DNA. The procedure as described by Staron et al. (1) seems satisfactory, provided that the absorbances are measured at 470 m,u, and the corresponding DNA concentrations are read from a calibration curve. In addition, the volume as well as TCA concentration of the samples and the DNA standard with which they are compared, have to be rigorously standardized. Neither heating of the DNA in 5% TCA nor addition of TCA to the sample was found to be essential to the reaction.

DETERMINATION

OF

DNA

WITH

321

HzS04

SUMMARY

A simple reaction described by Staron et al. (1) for the determination of DNA was studied, and found to be useful. A few modifications were introduced. ACKNOWLEDGMENT The author gratefu,lly Jenny C. Adams.

acknowledgea

the expert

technical

assistance of Mrs.

REFERENCES 1.

STARON,

T.,

ALLARD,

C.,

AND

CEUMBRE,

M., Compt.

Rend.

Acad.

Sci.

254,

765

(1962). Z., in “The Nucleic Acids” (E. Chargaff and J. N. Davidson, 1, p. 2%. Academic Press, New York, 1955. 3. SCHMIDT, G., AND THANNIJAUSP;R, S. T., J. Biol. Chem. 161, 83 (1945). 4. SEYDEL, J. K., AND GARRETIT,E. R., Anal. Chem. 37, 271 (1965). 5. SCHNEIDER, W. C., J. BioE. Chem. 164, 747 (1946). 2. DISCHE,

eds.), Vol.