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 . 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 . Chromatographic methods, especially HPLC , 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  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),
The resulting product, mono-thiol,
In 1987 we had a project including reversible modification of SH-group in NADPH-cytochrome P-450 reductase. We decided to get a paramagnetic analogue of the Ellman reagent, stable nitroxyl biradical, containing disulfide bond . 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, , 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))
This approach combines advantages of the methodology developed by Ellman  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,.
The observed ESR spectrum of.
In the presence of a free thiol group the reaction of thiol-disulfide exchange takes place:
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.
Figure 2 shows the effect of reduced glutathione, GSH, on the ESR spectrum of.
Note that the integral intensity of the ESR spectrum of.
The proposed methodology allowed quantitative assessment of glutathione concentrations in mouse erythrocytes , in hamster ovary cells [10,14 ] and various types of malignant cells [15,16]..
In contrast to conventional methods, our approach is non-invasive and suitable for work with
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
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 .
3. Determination of availability of Thiol groups in proteins
Traditionally, both alkylation and acylation spin labels have been used for chemical modification of proteins using SNRs. After incubation of nitroxyl radical with protein, the modified protein is separated from the free SNR by gel filtration, dialysis or precipitation. The use of biradical.
3.1. ESR study of the alcohol dehydrogenase free SH groups
Figure 3 illustrates the kinetics of chemical modification of the thermophilic alcohol dehydrogenase
3.2. ESR study of the alliinase’s SH groups
Alliinase (Cys sulfoxide lyase, alliin lyase, C-S lyase; EC 188.8.131.52) from garlic (Allium sativum) is an enzyme that uses pyridoxal-5’-phosphate (PLP) as a cofactor to catalyze the conversion of a nonprotein amino acid alliin, Sally cysteine Sulfoxide, to allicin (diallyl thiosulfinate), pyruvate, and ammonia, as shown in the following scheme:
Allicin, a product of the enzymatic reaction of alliinase with alliin, is a well-characterized, biologically active compound of garlic. It is responsible for the pungent odor and for a variety of biological effects attributed to garlic preparations, including antimicrobial, anticancer, antiatherogenic, and other activities [23, 24]
Incubation of native alliinase either with 4,4’-dithiodipyridine (DTP) or with 5,5’-dithio-bis-(2-nitrobenzoic acid) (Ellman reagent) in the presence of 6M guanidine- HCl provided evidence for the existence of two free cysteine residues in the alliinase molecule.
To identify the free cysteine residues, alliinase was modified by treatment with N-(4-dimethylamino-3, 5-dinitrophenyl) maleimide (DDPM) and digested with trypsin, chymotrypsin or pepsin . Peptides in digests containing the nitrophenyl chromophore were separated and detected on a 360- nm absorbance profile using reversed-phase HPLC. By analyzing the trypsin and chymotrypsin digests, we were able to identify a single (but different) Cys-containing peptide in each case. In the case of trypsin, it was a peptide containing a sequence with Cys220, and in the case of chymotrypsin the peptide contained the sequence with Cys350 . Treatment with pepsin made it possible to identify both of these free cysteine residues simultaneously in one digest. These experimental findings (predating the alliinase structure determination) provided direct confirmation that the two free thiols in the alliinase molecule .
Using ESR spectroscopy, we examined the availability of the free —SH groups of alliinase for chemical modification with the disulfide containing biradical.
Figure 5 shows the increase in peak intensity of the ESR signal for the reaction between the biradical and the native alliinase. These data demonstrate that the kinetics of modification occur at two different rates. Pretreatment of alliinase with
As shown earlier with biochemical methods, the enzyme subunit contains two free thiols: Cys220 in the PLP-binding domain 2 and Cys350 in the C-terminal part of domain 1 (C7 and C8, respectively) (Figs7 and 8) located relatively far from the active site and from the substrate-binding area. As shown in Figure 8(A), Cys220 is located on the surface of the alliinase molecule, while Cys350 is in a more buried location but is still water-accessible. The free thiol groups of Cys220 and Cys350 have different relative orientations with respect to each other (Fig. 8B), which might affect their chemical modification rates by.
4. Reversible modification of Thiol groups in proteins
Free thiol groups, whether intrinsic or introduced by site-directed mutagenesis are convenient targets for introduction of stable nitroxyl radicals, SNRs, into proteins. Now, this approach, named site directed spin labeling is very popular, because it can give information about structure (mobility) of different parts of the protein globule [31-33]. “Classical” SNRs used for modification of thiol groups, such as NR-labeled derivatives of iodoacetamide and maleimide, yield strong covalent S-C bonds which do not permit release of the spin label from the protein. Chemical modification using a disulfide-containing SNRs permits subsequent demodification by a low-molecular weight thiol such as mercaptoethanol, reduced glutathione, cysteine or dithiothreitol. Such demodification, performed in conjunction with simultaneous measurements of activity and of structural characteristics, allows evaluation of the contribution of the group modified to the stability and 3D structure of the protein studied. Berliner et al.  used the spin label MTSSL, for reversible chemical modification of Cys 25 in papain. We made use of biradical.
4.1. Acetylcholinesterase from
Torpedo Californica ( TcAChE)
Cys 231, a deeply buried residue in
Demodification of spin labeled protein by GSH (see eq.5), with concomitant release of the free monoradical spin label, done by ESR control, did not result in recovery of enzymatic activity. The use of a wide repertoire of physicochemical and biochemical techniques subsequently established that both the modified and demodified enzymes had assumed a partially unfolded, molten globule,
4.2. TBADH and NADPH-cytochrome P-450 reductase
However, in both cases removal of the bound spin label by treatment with the free thiol according to eq,5, resulted in immediate reactivation (Fig. 9). Spectroscopic measurements showed that modification had changed neither the tertiary nor secondary structure of the proteins and could be protected by affine inhibitor NADP+.
4.3. Alliinase from garlic (
We have shown recently that modification of Cys 220 and Cys 350 of alliinase with.RS-SR. does not change its enzymatic activity . In this case chemical modification of both free cysteine residues was found to leave both the secondary and the tertiary structure of the enzyme unchanged. This might be attributable to the marked thermodynamic and structural stability of alliinase, as well as the relatively long distances from modified free cysteines to the active center of the enzyme (see fig 7). This experimental finding permits one to use cysteines of alliinase for covalent binding with antibodies for targeted delivery of enzyme and for site-specific allicin generation to inhibit cancer cells proliferation [44,45].
For quantitate determination of sulfhydryl groups in low molecular weight compounds and proteins the symmetrical biradical containing disulfide bond, bis(2,2,5,5-tetramethyl-3-imidazoline-1-oxyl-4-il)-disulfide, or.
Different from the traditional spin label method, the.
Dedicated to the memory of Leonid Volodarsky
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