**1. Introduction**

Sulfhydryl (thiol, SH) groups of proteins and of low-molecular weight compounds, such as glutathione (GSH) and cysteine (Cys) play important roles in numerous biological processes. In the last decades, interest in the redox state of SH groups in proteins has grown because thiol-disulfide exchange has been found to play an important role in protein folding and to influence protein stability (1-3).

In cells, the ratio/equilibrium between oxidized and reduced forms of glutathione and between cysteine and cystine (main cells antioxidants) affect thiol balance and redox status of cells and proteins (4). In general, spectroscopic and chromatographic methods are used for quantitative determination of low-molecular thiols and sulfhydryl groups in proteins. Optical methods are employed to detect absorption or fluorescence, which appears on results of interaction between reagents and free SH groups. However, samples must be optically transparent, so preliminary homogenation and centrifugation of biological samples and other procedures are necessary (4). Chromatographic methods, especially HPLC (4), cannot be used for express analysis of thiol status of biological samples.

Among the optical methods for determining the free thiol groups the method proposed by Ellman (5) is definitely in the first place. This approach is based on the thiol-disulfide exchange reaction between the disulfide containing reagent (5,5'-dithiobis-(2-nitrobenzoic acid, DTNB), **ES-SE**, and the free thiol, **SH-T**:

$$\text{ES-SE} + \text{SH-T} \Rightarrow \text{ES-ST} + \text{ES} \cdot \tag{1}$$

The resulting product, mono-thiol, **ES- ,** has a characteristic optical spectrum (**λmax**= 412 nm) with a known extinction coefficient, **ε**=14 150 M-1cm-1. It is the simplicity of this method that has determined its widespread use (more than 13,500 citations for 50 years!). However, this

method suffers from all the drawbacks typical for other optical methods: the impossibility of measuring in a colored, scattering, and turbid media, i.e. in real biological systems. In addition, the sensitivity of this method is often insufficient.

In 1987 we had a project including reversible modification of SH-group in NADPHcytochrome P-450 reductase. We decided to get a paramagnetic analogue of the Ellman reagent, stable nitroxyl biradical, containing disulfide bond (6). We hoped that, if successful, the biradical would enter the free thiol/biradical thiol-disulfide exchange reaction, which could be followed by ESR. Our colleagues, Vladimir Martin and Tatyana Berezina from the team of Prof. Leonid Volodarsky (Institute of Organic Chemistry, Novosibirsk), synthesized biradical for the task (6,7)

In contrast to the known at that time disulfide containing spin label, [(1-Oxyl-2,2,5,5 tetramethylpyrroline-3-methyl) methanethiosulfonate],MTSSL, (8), our biradical allowed us to kill two birds with one stone: (a) to measure the kinetics of chemical modification of available SH groups in the protein (by appearance of free radical in solutions) and (b) using a traditional technique, i.e. gel- filtration or dialysis, to get the spin-labeled protein after incubation with our probe.(See eq.2 and Fig. 1))

**Figure 1.** ESR spectra of biradical and its reaction products. (**A) –** the monoradical formed on a reaction with low-molecular weight thiol or free SH group in the protein and (**B) -** the immobilized radical formed on a reaction with protein-linked SH group.

This approach combines advantages of the methodology developed by Ellman (5) that makes use of thiol – disulfide exchange reaction (see eq. 1) and of ESR, that provides high sensitivity and possibility of carrying out work in colored and/or turbid and scattering media, such as cells, tissue culture, blood, etc.

### **2. Use of SNRs for determination of Thiol status in cells**

For this purpose the symmetrical biradical containing disulfide bond, bis(2,2,5,5 tetramethyl-3-imidazoline-1-oxyl-4-il)-disulfide, **. RS-SR.** was synthesized:

biradical for the task (6,7)

addition, the sensitivity of this method is often insufficient.

incubation with our probe.(See eq.2 and Fig. 1))

formed on a reaction with protein-linked SH group.

media, such as cells, tissue culture, blood, etc.

tetramethyl-3-imidazoline-1-oxyl-4-il)-disulfide, **.**

**2. Use of SNRs for determination of Thiol status in cells** 

method suffers from all the drawbacks typical for other optical methods: the impossibility of measuring in a colored, scattering, and turbid media, i.e. in real biological systems. In

In 1987 we had a project including reversible modification of SH-group in NADPHcytochrome P-450 reductase. We decided to get a paramagnetic analogue of the Ellman reagent, stable nitroxyl biradical, containing disulfide bond (6). We hoped that, if successful, the biradical would enter the free thiol/biradical thiol-disulfide exchange reaction, which could be followed by ESR. Our colleagues, Vladimir Martin and Tatyana Berezina from the team of Prof. Leonid Volodarsky (Institute of Organic Chemistry, Novosibirsk), synthesized

In contrast to the known at that time disulfide containing spin label, [(1-Oxyl-2,2,5,5 tetramethylpyrroline-3-methyl) methanethiosulfonate],MTSSL, (8), our biradical allowed us to kill two birds with one stone: (a) to measure the kinetics of chemical modification of available SH groups in the protein (by appearance of free radical in solutions) and (b) using a traditional technique, i.e. gel- filtration or dialysis, to get the spin-labeled protein after

**Figure 1.** ESR spectra of biradical and its reaction products. (**A) –** the monoradical formed on a reaction with low-molecular weight thiol or free SH group in the protein and (**B) -** the immobilized radical

This approach combines advantages of the methodology developed by Ellman (5) that makes use of thiol – disulfide exchange reaction (see eq. 1) and of ESR, that provides high sensitivity and possibility of carrying out work in colored and/or turbid and scattering

For this purpose the symmetrical biradical containing disulfide bond, bis(2,2,5,5-

**RS-SR.**

was synthesized:

The observed ESR spectrum of **. RS-SR.** is typical for symmetrical biradical with intermediate character of exchange between two unpaired electrons (9) (see fig.2A).

In the presence of a free thiol group the reaction of thiol-disulfide exchange takes place:

$$\cdot \text{RS} \cdot \text{-SR} \cdot + \text{HS} \cdot \text{-A} \xrightleftharpoons \text{RS} \cdot \text{SA} \cdot \text{+R} \cdot \text{-SH} \tag{2}$$

$$\cdot \text{RS} \cdot \text{-SA} + \text{HS} \cdot \text{A} \rightleftharpoons \text{AS} \cdot \text{-SA} + \text{\textbullet R} \cdot \text{-SH} \tag{3}$$

**Figure 2.** The effect of GSH on ESR spectra of **. RS-SR.** (100 μM) in PBS, pH=7.5. Spectra **A, B, C** were carried out at: *gain 5x104*, modulation amplitude 1 G, microwave power 10 mW; Spectrum **D**- *gain 3.2x103***,** modulation amplitude 1 G, microwave power 10 mW.

The exchange integral, J, was estimated: J = 3.6 aN (9,10). The absence of any change in the ESR spectrum up to 80oC can be interpreted in terms of existence of a single average conformation of **. RS-SR.** in solution.

Figure 2 shows the effect of reduced glutathione, GSH, on the ESR spectrum of **. RS-SR.** : with increasing GSH concentration, the biradical spectral components (1,2,3,4,5,6,7,8,9) decrease with simultaneous increase of monoradical components (1',2'3'). Thus nine broadened components of biradical decrease with concomitant appearance of three narrow components of two monoradicals as a result of the sequential reactions:

Note that the integral intensity of the ESR spectrum of **. RS-SR.** remains unchanged. The peak intensities of the monoradical components **(. R-SH)** resulting from reactions (2,3) are about 17-fold higher than those of the corresponding biradical components (whose position in the field coincides with that of monoradicals). These phenomena provide the physical basis for the proposed method.

Four reviews give a detailed description of the physical and chemical background for the practical use of the biradical **. RS-SR.** for thiols meaurements (10-13).

The proposed methodology allowed quantitative assessment of glutathione concentrations in mouse erythrocytes (7), in hamster ovary cells (10,14 ) and various types of malignant cells (15,16). **. RS-SR.** is a hydrophobic molecule and , therefore, can easily cross biological membranes and penetrate into cells.

In contrast to conventional methods, our approach is non-invasive and suitable for work with *intact* cells and tissues. It is also extremely sensitive, permitting determination of GSH concentrations in as few as 100 cells (11,14). The method was also used successfully to measure GSH in an isolated reperfused heart (11). Using the biradical **. RS-SR.** in conjunction with the spin trap DMPO, we were able to demonstrate that the efficacy of oxygen radical generation, stimulated by redox active quinones, correlated with GSH levels and the induction of expression of GSH transferase in cancer cells (15,16). The biradical method was also successfully applied to the monitoring of GSH levels in cancer cells treated with allicine, an active component of garlic, which can arrest the proliferation of cancer cells (17).

The biradical method was also used for direct determination of the catalytic activity of acetylcholinesterase in homogenates of the heads of individual larvae of the bollworm *Heliothis armigera,* following the rate of hydrolysis of acetylthiocholine by monitoring reduction of biradical by the thiocholine produced , according to eq.2(10,18).

Note that synthesis of new disulfide containing SNR is still in progress (19,20). Using the new disulfide containing biradical, the glutathione level (by L-band ESR spectrometer) in tumors in nude mice was measured. This "improved" biradical contains N-15 where deuterium substitutes for hydrogen atoms. This approach enhances the method sensitivity (20).
