EPR method for the measurement of cellular sulfhydryl groups

EPR method for the measurement of cellular sulfhydryl groups

Volume 290, number I,& 243-246 FEBS 10219 0 1991 Federation of European Biochemical Societies 00145793/91/$3..50 EM. Weiner’, H. Hu2 and H.M. Sept...

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Volume 290, number I,& 243-246 FEBS 10219 0 1991 Federation of European Biochemical Societies 00145793/91/$3..50

EM.

Weiner’,

H. Hu2 and H.M.

September

1991

Swartz*

‘Chemkal Physics Department, Weizmann Institute of Science, Rehovot, Israel 76100 and ZUniversity of r/linois, College of Medicine and EPR Research Center, 190 Medical Sciences Building, 506 S. Muthews, Urbana, IL 61801, USA Received 16 July 1991 .An EPR method that can measure the concentration of sulfhydryl groups in intact cells has been developed using a specially designed stable nitroxyl biradical. The biradical, AS-Sli, contains a disulfide bond and readily undergoes thiol-disulfide exchange reactions with thiols resulting in a characteristic EPR spectrum which can be analyzed to provide a quantitative measure ofsulfhydryl groups. The data obtained from the EPR method are in good agreement with those obtained from the conventional optical method using Ellman’s reagent. The advantages of the EPR method are that the measurement can be carried out on intact cells or any other highly colored. absorbing and/or scattering solutions and the sensitivity is such that only a few cells (- 100) are needed for each quantitative measurement. Nitroxyl biradical; Thiol-disultide

exchange; EPR; Intact cell; Glutathione

1. INTRODUCTION Glutathione (GSH), a natural tripeptide, occurs in all mammalian tissues at high concentration (I-5 mM) (see e.g. [I]), primarily in its reduced form. It is the main SH-containing, low molecular weight component of living tissues. Of the numerous biological functions of GSH [2,3], the two most important functions are: removing toxic metabolites, including peroxides, from the cell and maintaining cellular sulfhydryl groups in their reduced form. Many abnormalities and diseases are due to changes in GSW levels [2,3]. In this connection the quantitative determination of GSH in living tissues is one of the most important methodical and scientific problems. Optical and chromatographic methods have been used to determine the SH groups quantitatively [4] but there are disadvantages associated with these methods. The optical methods require that the sample be optically transparent so they are difficult to apply with highly scattering, absorbing or colored systems such as cell suspensions. Chromatographic methods are rather labor-consuming and are not well-suited to rapid analysis. The proposed NMR method is not sensitive enough for many uses [5]. Recently we have proposed a new method for the quantitative measurement of SH groups in low- and high-molecular weight compounds based on EPR spectroscopy [6]. The method is based on thiol-disulfide exchange reactions of a stable nitroxide biradical containing a disulfide bond, &S-SA (Fig. l), with SH groups of thiols. This method has been used to deterCorresportdenceaddress: L.M. Weiner, Chemical Physics Department, Wcizmann Institute of Science, Rehovot, Israel 76100.

Published by Elm&r Science Publishers B. V.

content

mine concentrations of cysteine and GSH in the blood of mice and rats [6] and for measuring acetylcholinesterase activity in homogenate obtained from insects [7]. In this paper we report on the extension of the EPR method to cells, measuring GSH in intact Chinese hamster ovary (CHO) cells. The proposed method is shown to be consistent, within experimental error, with the method using the Ellman reagent [g]. Quantitative measurement of GSH could be made with only 100 CNO cells.

2. MATERIALS

AND METHODS

l&-Sli was synthesized as described in [6]. Since l&--S~ is hydrophobic, it was dissolved in DMSO and added to the sample with the final concentration of DMSO less than 5%. The CHO cell line was a gift from Dr L. Hopwood, Medical College of Wisconsin and has been maintained in our laboratory for several years. The cells were grown to confluence at 37°C in McCoy’s 5a medium and collcctcd by trypsination. The cells (2 x 105/ml) were then incubated at 37°C for 24 h in a Bellco spinner flask just prior to their use in these experiments. For the routine mcasurcment of SH groups in intact cells, 2.5 x 10” cells were suspended in 1.5 ml phosphate buffer solution containing 8 x IO-’ M k&Sk and the EPR spectrum was taken after 3 min. For measurements on frozen-thawed cells, 2.5 x10” cells were frozenthawed 3 times, then 8 xIO-~ M l&-Sk was added and the EPR spectrum was taken after 3 min. For the measurement of SH groups in the aqueous compartment of cells, frozen-thawed cell suspensions were centrifuged at 6000 x g then 8 x IO-’ M l&-Sk wus added to the supernatant. In each EPR measurement, a small portion (100/11) ofeach preparation was placed into a gas-permeableTeflon tube (Zeus Industries, Raritan, NJ), 1 mm in diameter, 10 cm long, folded in half and placed in a quartz tube open on both ends, the quartz tube was placed in the horizontal EPR cavity. For measurement of the total SH groups in CHO cells, 2.5 X 10” cells were freeze-thawed 3 times and 0.5 mM Ellman’s reagent was added to reach a total volume I .5 ml. The supcrnatant was taken after

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centrifugation at 6000 x gand the optical density was measured at 412 nm. For measurement of SH groups in the aqueous cellular compartment, Ellman’s reagent was added to the supernatant after the centrifugation and the optical density of 412 nm was measured (E~,~= 1.4 x 104 M-‘.cm-’ [9]). Cellular St-l groups also were measured by both EPR and optical methods after incubation ofCH0 cells with L-buthionine-S,R-sulfoximine (BSO) (purchased from Chemical Dynamics), which inhibits glutatione synthesis [IO]. 2.5 x IO6cells in I ml medium were incubated with I mM BSO at 37°C for 7 h and washed 3 times with phosphate buffer solution; then the cellular SH groups were measured by the methods described above. All EPR spectra were taken at room temperature (22°C) on a Varian E-109 spectrometer equipped with an EPR data acquisition system [I I]. The usual instrumental parameters were: microwave frequency, 9.05 GHz; incident microwave power. 5 mW: center of the field. 3210 G; scan range. 100 G; and field modulation, 1 G. Optical measurements were taken at room temperature (22°C) on a Hewlett Packard 8452A Diode Array Spectrophotometer equipped with data acquisition system. The test of sensitivity of the EPR method was done in a flat quartz cell to achieve maximal sensitivity. Concentration of both cells and ri,S-.Sfi were gradually decreased to the levels where the EIBR signal and iCschange due to reactions between cellular SH groups and the biradicals could be observed at a signal-to-noise ratio around 4: I. A receiver gain of IO’, modulation amplitude of 4 G and incident microwave power of 50 mW were used to optimize the signal.

J.

RESUETS

AND DISCUSSION

The biradical &&-SR participates in thiol-disulfide exchange reactions with thiols resulting in the change of its EPR spectrum because of the production of the monoradicals, d-SH and BS-SR [6,7]: riS-S&-e

B-SH;R-SH+BS-SR r

(1)

The difference in the EPR spectra of RS-SR, R-SH and BS-SR forms the basis for the EPR method (Fig. 2). For [&S-S&] > [B-SH) the equilibrium in reaction (1) is shifted to the right-hand side and an increase in EPR signal intensity of the monoradicals formed can be used for the quantitative determination of GSH in the sample. As has been shown in [6] an increase in signal intensity of a component of the spectrum due to the monoradical is approximately 16 times greater than a decrease in the signal intensity of component of the EPR spectrum due to biradical. The double-integral of the spectrum remains unchanged [6]. Therefore, measurement of the relative increase of the peak height of the monoradical component upon addition of SI-l containing low molecular weight compounds to the system is

September

1991

(A)

Fig. 2. Change SH. Spectrum buffer solution after 1.6 x IO“

of the EPR spectrum of d??-sd after its reaction with alone in phosphate (pH 7.3) and spectrum B was from the same solution WIGSH was added. The EPR instrumental parameters were as described in section 2.

A was from 8 x 10e5 M dS-Sd

a very sensitive method for the determination of their absolute concentration. Fig. 3 shows the relationship between the EPR parameter C, defined in Fig. 2, and the concentration of sulfhydryl groups of glutathione. We have found experimentally that the linear dependence of the parameter C on concentration of GSl-I is retained up to the ratio [rZS-Sri]l[CSr?r] = 3. As this ratio decreases (the concentration of GSH increases) the dependence of (I - Z,)l& deviates from linearity, which reflects the influence of the back reaction (K,) in thiol-disulfide exchange (see Eqn. 1). As is clearly seen in Fig. 3, at a fixed concentration [AS-SR[ (in our case 8 x IO-” M) GSI-I can be reliably measured within the range l-25 PM. In addition, no reduction of RS-SR by GSH was found. Table I shows the results of the comparative study in which the SH groups associated with CWO cells were measured by both EPR and optical methods after different treatments of the cells. The results from the two methods are in good agreement. It is obvious that one of the advantages of the EPR method is that it could be applied to an intact cell suspension or any other highly colored, absorbing or scattering system. The data from the intact cells by EPR are not statistically different

Table I Mcasurcmcnt of cellular SH groups by EPR and optical methods Unit: IO” SH groups/cell + SD (number of experiments)

Fig. I. Chemical structure of the bitadical

244

Rk-SR:

Treatment

EPR method

Optical method

Intact cells Freeze-thawed cells Supernatant after freeze-thawing

4.8 + I.4 (4) 4.0 +I 0.7 (4) 3.2 & 0.2 (4)

_*.-----__4.7 & 0.3 (2) 3,6 t 0.4 (2)

FEBS LETTERS

Volume 290,number I,2

g I2

September 1991

1.2~l.O--

d 0.8-iz ir 0.6.. f!3 0.4..

5

CONCENTRATION

20

OF GLUTATHIONE

~JJU)

Fig. 3. Effect of GSH on the empirical EPR parameter C of biradical /?S-Sd. GSH was added gradually to the phosphate buffer (pH 7.3) which contained 8 x10-’ /&-S&and then an EPR spectrum was taken at each given concentration of GSH. The EPR instrumental parameters were as described in section 2.

Fig. 4. Test of sensitivity of the EPR method. The spectra were taken from a tlat cell which contained 0.25 x low6 M RS-SR in phosphate buffer (pH 7.3). Spectrum A was taken without cells and spectrum B was taken with 130 CHO cells in the flat cell. The setting receiver gain IO’; modulation 4 Gauss; incident microwaver power 50 mW; these settings were selected to increase the signal-to-noise ratio. The other EPR parameters were as described in section 2.

from data from the frozen-thawed cells, by both EPR and optical methods, which indicates that the biradical [email protected] readily penetrated into the cells: this was expetted because it is uncharged. The data by both EPR and optical methods from the supernatants after freezing-thawing the cells indicate that the SH groups associated with cell membranes and membrane proteins contributed 20-30% to the total cellular SH groups measured on both the intact and frozen-thawed cells. To determine whether a decrease in cellular GSH level would give similar results with the two methods, we employed BSO, which inhibits GSW biosynthesis and reduces the level of GSH in cells (Table II). Again the results from the two methods were in good agreement; the total cellular SI-I groups were reduced by 20-30% and the results were reproducible. The cells were counted both before and after incubations with BSO and no changes in cell numbers were found. To estimate the abolute sensitivity of the method we used a minimal concentration of &S-SR and recorded EPR spectra using a flat cell configuration (Fig. 4). Spectrum A is from 0.25 @l &S-S-R alone in buffer and spectrum B is taken after about 130 cells are added. Despite the signal-to-noise ratio of about 4:l in spectrum A, on addition of the cells an increase in peak height of the monoradical component is reproduced

reliably. Using the calibration curve, we have estimated the c;ontent of GSH in the sample to be about O.O27yM, so the estimated cellular concentration 0fGSI-I is about 5 mM. A potential limitation of the use of nitroxide for quantitative measurements“in ceils and tisrhes is their possible reduction to diamagnetic hydoxylamines [ 12,131. We found, however, that under our usual conditions (2.5 x 10’ CI-IQ cells in 1.5 ml medium and 8 x lo-’ M i&!-S&), on.1.yabout 4% of J&-Sri was reduced after 5 min, which would not affect the measurement of cellular SH groups by the EPR method. In summary, the EPR method developed here for the measurement of cellular SH groups produced data consistent with those obtained by optical method. The advantages of the EPR methods are that the measurements can be carried out on intact cells or any other highly colored, absorbing and scattering solutions and only a few cells (- 100) are needed in each measurement. This method opens up the possibility of determining GSH concentrations in individual cell compartments, the occurrence of such changes have been suggested in some pathological conditions such as Parkinson’s disease /14]. Acltnolvlerlgettrenrs:

This research received financial support from the Israel Ministry of Absorption.

Table II Measurement of cellular SH groups after incubation with BSO Unit: IO9 SH groups/cell i SD (number of experiments) Trcatmcnt* Control Incubated with I mM BSO for 7 h *Cells were freeze-thawed

EPR method

Optical method

4.2 + SD 0.3 (2) 3.5 -e SD 0,l (2) 2.6 +ZSD 0,l (2) 2.9 + SD 0.0 (2)

three times prior to the measurement.

REFERENCES [I] Metzler, D.E. (1977) Biochemistry (Chapter 7), Academic Press, New York. [2] Dolphin, A., Poulson, R. and Avramovic, 0. (1989) Gluthatione: Chemical, Biochemical and Medical Aspects (Part A), Wiley, New York. [3] Meister, A. and Anderson, M.E. (1983) Annu. Rev, Biochem. 52, 71 I-760. [4] Jacoby, W.B. and Griffith (1987) Methods in Enzymology (Vol. 143), Academic Press, San Diego.

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151Rabenstein,

D.D., Reid, R.S., Yamashita. G., Tan, KS. and Alan. P.A. (1986) Anal. Chem. 58, 1266-1269. WI Khramtsov, V.V., Yelinova, V.I., Weiner, L.M., Berezina, T.A., Martin, V.V. and Volodarsky, LB. (1989) Anal. Biochem. Igt, 5%63. 191 Weiner, L.M. and Khramtsov, V.V. (1990) Proceedings of XPV International Conference of Magnetic Resonance in Biological Systems, pp. I S-44, University of Warwick. Boyne, A.F. and Ellman, G. (1992) Anal. Biochem. 46,639-6.53. Collier. RN. (1993) Anal. Biochcm. 56, 310-311.

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[IO] Russo, A., DeGraff, W., Friedman, N. and Mitchell, 3.8. (1986) Cancer Res. 46.2544-2848. [I l] Morse, P.D. II (1989) Biophys. J. 51, 440a. [la] Keana. J.F., Pou, S. and Rosen, G.M. (1989) Magn. Resort. Med. 5, 525-536. [13] Chen, K., Morse, P.D. II and Swartz, H.M. (1988) liochim. Biophys. Acta 943,477484. [I41 Cohen, G. and Spina, MB. (1988) in: Progress in Parkinson Research (Hefti, F. and Weiner, W.J., eds.) pp. 119-126, Plenum, Now York.