**5. Conclusions**

380 Nitroxides – Theory, Experiment and Applications

**Figure 9.** Influence of NADP+ and DTT on effect of biradical **.**

introduced at 10 min (shown by arrow). (from ref. 21)

incubated with the biradical without (closed circles) and with (open circles) NADP+. DTT was

and for site-specific allicin generation to inhibit cancer cells proliferation (44,45).

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

**Figure 10.** ESR spectrum of Alliinase-radical conjugate. Protein concentration was 13 μM in 10 mM PBS buffer, pH7.6. **(A) -** ESR spectrum of modified protein, ESR conditions: microwave power 10 mW; modulation amplitude 1 G; gain 2x105. **(B)**- ESR spectrum of sample **(A)** 4 min after addition of 0.2 mM

of glutathione. ESR conditions were the same as in **(A)**, but the gain was 3.2x104.

**RS-SR.** on TBADH activity. Enzyme was

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 **. RS-SR.** , was synthesized. The biradical, **. RS-SR.** permits quantitative determination of glutathione and cysteine in biological systems ( 'thiol status') by use of ESR. Note that any *routine* CW ESR spectrometer can be used for the measurements! This non-invasive express method has very high sensitivity: possibility of measuring < 10-12 mol of SH groups in a sample. Thus, concentrations of glutathione and cysteine in living cells were determined under physiological conditions, as well as in cells, including cancer cells, treated by pro-and anti-oxidants.

Different from the traditional spin label method, the **. RS-SR.** usage makes it possible to assess the rate of modification of SH groups in proteins (availability) and the influence of substrates, inhibitors, coenzymes, other proteins, artificial and biological membranes, pH, etc. in direct experiments. Modification of SH groups in proteins by **. RS-SR.** is *reversible*, which permits application of this approach, coupled with site directed spin labeling, for evaluation of stability and unfolding of proteins and different parts of the protein globule.
