ANALYTICAL BIOCHEMISTRY 28, 369-375 (1969)

Estimation of Thiol Content of Basic Proteins in Acid

Solution by Saville’s Method

PAUL TODD l AND MICHAEL GRONOW 2

Depa&nmt of Biochemistry, University of Oxford, Oxford, England

Received September 12,1968

As a result of the possible regulatory role of the basic nuclear proteins (histones), the histones have received considerable atten- tion in recent years (see ref. 1). However, the method by which the histone to DNA association is disturbed sufficiently to allow transcription of the DNA to occur is, as yet, unknown. Various theories have been put forward, and of particular interest is the work of Ord and Stocken on the importance of thiol (-SH) groups in the metabolism and function of certain histone fractions (2-5). The relatively small amount of -SH present in these proteins means that a very sensitive and specific method of estimation must be used. Although many -SH estimations are performed at neutral or slightly alkaline pH (e.g., ref. 6) these methods are often not applicable to the histones, owing to the ease of aggregation of these proteins at about pH 5 (7). Therefore a method was sought that could be performed directly at acid pH.

It had previously been found (8) that the method of Saville (9) was ideally suited for the measurement of the nonprotein (or acid- soluble) -SH content of living cells and biological fluids. However proteins were not considered to be interfering substances (9) and therefore because of the sensitivity and specificity of the reaction might be applied to histone extracts.

The basis of this method is the ease of reaction of thiol groups at low pH with nitrous acid to give -S-nitroso derivatives. After removal of excess nitrous acid with ammonium sulfamate the -S- nitroso derivative is catalytically broken down by mercuric ions to give nitrous acid again. The amount of this acid liberated can be ascertained by adding sulfanilamide. This gives a diazo compound

1 Present address: Department of Biophysics, The Pennsylvania State Uni- versity, University Park, Pennsylvania 16802.

2 Present address: Department of Experimental Pathology and Cancer Re- search, University of Leeds, Woodhouse Lane, Leeds 2, England.

370 TODD AND GRONOW

which, on coupling with N-1-naphthylethylenediamine yields an in- tensely colored magenta dye with an absorption maximum near 650 nm, the absorption of which is linearly related to the original thiol concentration.

The sensitivity and specificity of this method make it ideally suited for the measurement of the nonprotein (or acid-soluble) sulfhydryl content of living cells and tissues and biological fluids (8,10). Because of the acid solubility and low thiol content of his- tones it appears that Saville’s method should be suitable for the estimation of sulfhydryl groups of these basic proteins. This com- munication describes and evaluates a micro method, based on that of Saville, for the estimation of thiol groups in basic proteins, using histones as an example.

MATERIALS AND METHODS

Reagent-grade chemicals were obtained from May and Baker Co., Hopkins and Williams, Sigma (London) Chemical Co., or British Drug Houses, Ltd., N-1-naphthylethylenediamine dihydrochloride from Koch-Light, and 5,5’-dithio (2nitrobenzoic acid) from Aldrich Chemical Co. Inc., Milwaukee.

Histone solutions were prepared from the nuclei of rat hepatoma cells (ascitic form of Hepatoma 223-a butter yellow induced tu- mor) maintained by serial intraperitoneal transplantation in albino male Wistar rats. Nuclei were isolated by glass pestle homogeniza- tion of osmotically shocked cells, as has been described (11). His- tones were obtained by doubly extracting nuclei with 0.25 M HCl after other nuclear proteins had been removed by 8 M urea extrac- tion. Histones were stored frozen in this solution at concentrations of 1030 mg/ml. Protein concentrations were estimated by the method of Lowry et al. (12).

PROCEDURES

For the estimation of thiol concentration by Saville’s method, the following stock solutions were prepared:

A. 0.12% (w/v) NaNO, in distilled H’,O. B. 1 M H,SO,. C. 1 volume of A plus 4 volumes of B. D. 1 $?$I (w/v) ammonium sulfamate in distilled H,O. E. 6.9% (w/v) sulfanilamide in 0.4 M HCl. F. 1.0% (w/v) HgCl, in 0.4 M HCl. G. 2 volumes of E plus 1 volume of F. H. 0.2 ~JJ (w/v) N-1-naphthylethylenediamine hydrochloride in

0.4 M HCl.

THIOL ESTIMATION 371

J. 0.0020/o (w/v) reduced glutathione in 0.25 N HCI, deaerated. Solutions C and G were prepared a few minutes before use. With

the exception of H and J all other solutions could be stored for weeks or months at room temperature. The micro procedure was carried out as follows: Histone or standard (solution J) samples were added in a total volume of 0.2 ml in 0.25 M HCl to 3 ml round- bottom glass tubes. To this was added 0.05 ml of solution C, and the mixture was allowed to stand 5 min at room temperature; then 0.02 ml of solution D was added with vigorous stirring with a Vor- tex mixer.

To this were then added, in sequence, 0.1 ml of solution G and 0.15 ml of solution H. Color development was allowed to take place at room temperature for 10 to 30 min before optical density was measured by transferring the samples to 0.3 ml spectrophotometer cuvets and reading against thiol-free reagent blanks at 550 nm in a Hilger-Watts spectrophotometer adapted for micro cell operation. The total reaction volume was thus 0.52 ml. Absorption spectra of the colored products were determined in a Beckman DK-IIa record- ing spectrophotometer.

For the estimation of thiol concentration by Ellman’s method (6)) the following stock solutions were prepared:

K. 0.4% (w/v), 5,5’-dithiobis (Znitrobenzoic acid) in 0.1 M Tris, pH 7.8.

L. 1 volume of K plus 9 volumes distilled water. M. 12M tris (hydroxymethylamino) methane, pH 7.8. N. Saturated urea in distilled water. P. 0.01% (w/v) reduced glutathione in deaerated 0.25 2M HCl. Solution L was prepared a few minutes before use; other solu-

tions were stored at 4’ for several days, except P, which was made up immediately before use.

Samples, standards, and blanks were neutralized and brought to a volume at 0.6 ml, including up to 0.4 ml of solution N if desired; to this were added, in sequence, 0.2 ml of solution M and 0.2 ml of solution L. Color was read in 1 ml cuvets at 412 nm in a Hnicam Sp600 spectrophotometer against thiol-free reagent blanks, The glutathione standard used under these conditions gave a molar ex- tinction coefficient of E41’ - 1.10 X 104. 1omm -

RESULTS

1. Sensitivity Range

By using reduced glutathione as test substance, it was determined that the micro adaptation of Saville’s method was canable of esti-

372 TODD AND GRONOW

mating 0.5 to 15 nmoles of thiol and was linear over this range, as shown in Figure 1. Suitable volume increases extend the method to larger quantities of thiol. Quantities less than 1 nmole were esti- mated with a standard deviation which was about 10% of the mean when duplicate readings were made. The resulting optical density corresponds to a molar extinction coefficient of 4.0 s 0.1 X lo* for glutathione, so that the method is 3 to 4 times as sensitive as the Ellman procedure.

2. Absorption Spectrum

Figure 2 illustrates the similarity of the absorption spectra of glutathione-containing and protein-containing reaction mixtures.

n-MOLES GSH/0.52 ml.

FIG. 1. Sensitivity of Saville thiol assay to glutathione.

450 500 550 600 l.nm

FIG. 2. Absorption spectrum of color product of diazotized p-sulfanilamide in presence of glutathione (upper curve) and histone (lower curve).

THIOL ESTIMATION 373

The absence of obvious differences suggests an absence of inter- fering colored products in the protein-containing mixture. The occurrence of the absorption maximum at 540 nm was a consistent finding and appears to differ from the 550 nm maximum quoted by Saville.

3. Dependence upon Protein Concentration

As the amount of histone in the reaction mixture was increased from 0.7 to 175 pg, a linear increase in optical density was found. By using Ellman’s method in urea, it was determined that the his- tone sample used contained 26 nmoles of thiol/mg protein. Figure 3 presents a plot of optical density vs. glutathione SH and vs. his- tone SH using the Saville micro method. The estimation method appears to be independent of protein concentration.

4. Evaluation of Interfering Substances

Because basic proteins have many free amino groups, it was con- sidered possible that nitrous acid was consumed in reactions other than the S-nitrosylation of the thiol group. If this were the case, more nitrous acid would be needed; however, when the nitrous acid concentration was tripled, no increase in optical density was found. This finding is consistent with the stoichiometric fact that a vast excess of nitrous acid is used in the reaction mixture.

If nonthiol reactions contributed to the nitrous acid concentra- tion after mercury ion treatment, color development would be ex- pected in the absence of Hg ions. The omission of Hg ions from a

. GLUTATHIONE

0 HISTONE

0 I 2 3 4 5 6 7

n-MOLES THIOL /0.52 ml.

FIG. 3. Dependence of color development on histone concentration in Saville thiol assay. Histone thiol on the abscissa is based on estimations with Ellman’s method.

374 TODD AND GRONOW

reaction mixture containing 120 pg of histone did not give a ma- genta dye immediately, thus showing the color development to be specific for thiol groups only. Approximately 40 hrs was required for full color development without Hg catalysis.

The accidental reduction of disulfide bonds could lead to serious misinterpretations of protein chemistry, so the reactivity of oxi- dized glutathione was studied. It was found that 75 nmoles of disul- fide resulted in a zero optical density of 550 nm, compared to 13 nmoles of thiol required for an optical density of unity. It is un- likely that protein dissulfides contribute to color development.

It was also found that 2 ,umoles of methionine gave no color de- velopment. Apparently, the test is also insensitive to thio esters (10). Because of evidence that histones sometimes contain nucleo- tides (13) t adenine was tested, and it was found that 0.2 pmole of adenine gave no optical density in the system described.

DISCUSSION

The method of Saville appears to be applicable to the measure- ment of the thiol content of basic proteins. The evidence gathered here suggests that the estimation is highly specific for thiol groups and that no substances present in cell extracts are likely to inter- fere. It also has the advantage of being performed directly at acid pH, which avoids the aggregation of histones. The method should prove useful in tissue culture studies for which only minute amounts of nuclear protein can be obtained, specially when varia- tions during the cell cycle are being studied.

ACKNOWLEDGMENTS

We thank Drs. L. A. Stocken and M. G. Ord for their interest in and sup- port of this work, the British Empire Cancer Campaign for a grant to M. Gronow, and the Eleanor Roosevelt Foundation and the International Union against Cancer for a Cancer Research Fellowship to P. Todd.

REFERENCES

1. “Histories-Their Role in the Transfer of Genetic Information” (CIBA Foundation Study Group No. 24). Churchill, London, 1966.

2. Oan, M. G., RAAF, J. H., SMIT, J. A., AND STOCKEN, L. A., Biochem. J. 95, 321 (1965).

3. ORD, M. G., AND STOCKEN, L. A., Biochem. J. 98,888 (1966). 4. HILTON, J., AND STOCKEN, L. A., Biochem. J. 100,21C (1966). 5. OR& M. G., AND STOCKEN, L. A., Biochem. J. 102,631 (1967). 6. ELLMAN, G. L., Arch. Biochem. Biophys. 82,70 (1959). 7. CRUFT, H. J., MAURITZEN, C. M., AND STEDMAN, E., Proc. Roy. SOC. (LOW

don) B249,24 (1958). 8. GRONOW, M., Intern. J. Radiation Biol. 9,123 (1965).

THIOL ESTIMATION 375

9. SAVILLE, B., Analyst 83,670 (1958). 10. FRASER, L. B., AND CATER, D. B., &it. J. Cancer 21,235 (1967). 11. GRONOW, M., Biochem. J. 109,25P (1968). 12. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J.,

J. Biol. Chem. 193,265 (1951). 13. BONNER, J., Science 159,47 (1968).