**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 **. RS-SR.** permits direct measurement of the rate of protein modification by the monitoring the appearance of the free monoradical, **. R-SH,** in solution, (Fig. 1,eq. 2) thereby providing "visible" information about the rate of thiol-disulfide reaction (eq. 2) and, consequently, about the availability of the thiol group in the protein.

#### **3.1. ESR study of the alcohol dehydrogenase free SH groups**

372 Nitroxides – Theory, Experiment and Applications

**RS-SR.**

basis for the proposed method.

practical use of the biradical **.**

**RS-SR.**

membranes and penetrate into cells.

cells (15,16). **.**

in solution.

Note that the integral intensity of the ESR spectrum of **.**

**RS-SR.**

measure GSH in an isolated reperfused heart (11). Using the biradical **.**

peak intensities of the monoradical components **(.**

conformation of **.**

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

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

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

Four reviews give a detailed description of the physical and chemical background for the

The proposed methodology allowed quantitative assessment of glutathione concentrations in mouse erythrocytes (7), in hamster ovary cells (10,14 ) and various types of malignant

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

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,

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

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

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

an active component of garlic, which can arrest the proliferation of cancer cells (17).

substitutes for hydrogen atoms. This approach enhances the method sensitivity (20).

reduction of biradical by the thiocholine produced , according to eq.2(10,18).

**3. Determination of availability of Thiol groups in proteins** 

for thiols meaurements (10-13).

is a hydrophobic molecule and , therefore, can easily cross biological

**RS-SR.**

**RS-SR.** :

remains unchanged. The

**R-SH)** resulting from reactions (2,3) are

**RS-SR.**

in conjunction

Figure 2 shows the effect of reduced glutathione, GSH, on the ESR spectrum of **.**

components of two monoradicals as a result of the sequential reactions:

Figure 3 illustrates the kinetics of chemical modification of the thermophilic alcohol dehydrogenase *from Thermoanaerobacter brockii* (TBADH) by biradical **. RS-SR.** (10, 21). The high reaction rate suggests that when modified, the free thiol group is highly accessible. Modification of TBAD by [2-14C] iodoacetic acid and identification of the labeled peptide indicated that the thiol group labeled was that of Cys 203. In the presence of coenzyme, NADP+, the rate of modification falls (Fig.3, line C) providing evidence that NADP+ interacts with Cys 203. The kinetics of chemical modification of TBADH after removal of Zn2+ from its active site by treatment with phenanthroline were almost two-fold higher than for the native enzyme (Fig.3, curve B). As follows from HPLC analysis of the radio-labeled peptide in apo-TBADH, Cys 37, which serves as a ligand for Zn2+ in the active site, is available for modification. Using apo-TBADH double-labeled at both Cys 203 and Cys 37 with biradical,

**Figure 3.** Kinetics of modification of TBADH by biradical **. RS-SR.** . The peak intensity of the monoradical component, **. R-SH** (released to solution as a result of reaction between biradical and TBADH (see eq.2)) was monitored. **A**- native TBAD; **B**-apo-TBAD; **C**-TBADH in the presence of NADP+; **D**-TBADH pretreated with *p-*chloromercury benzoate. (from ref. 21)

we were able to estimate the distance between radicals covalently bound at these two cysteines (r~ 10 Å). Later, our colleagues in Weizmann Institute obtained an X-ray structure of TBADH at 2.5 Å resolution (22). The 3D structure has revealed that Cys 203 is indeed a surface residue which is occluded by the coenzyme NADP+ (Fig.4)

**Figure 4.** Representation of the monomer of TBADH (PDB entry 1ykf). Individual residues are represented as spheres colored in yellow, the NADP+ cofactor and Cys 203 are colored in cyan (left) and green (right), respectively.

### **3.2. ESR study of the alliinase's SH groups**

Alliinase (Cys sulfoxide lyase, alliin lyase, C-S lyase; EC 4.4.1.4) 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:

#### **Scheme 1.**

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.

374 Nitroxides – Theory, Experiment and Applications

green (right), respectively.

**Scheme 1.**

**3.2. ESR study of the alliinase's SH groups** 

anticancer, antiatherogenic, and other activities (23, 24)

we were able to estimate the distance between radicals covalently bound at these two cysteines (r~ 10 Å). Later, our colleagues in Weizmann Institute obtained an X-ray structure of TBADH at 2.5 Å resolution (22). The 3D structure has revealed that Cys 203 is indeed a

**Figure 4.** Representation of the monomer of TBADH (PDB entry 1ykf). Individual residues are

thiosulfinate), pyruvate, and ammonia, as shown in the following scheme:

represented as spheres colored in yellow, the NADP+ cofactor and Cys 203 are colored in cyan (left) and

Alliinase (Cys sulfoxide lyase, alliin lyase, C-S lyase; EC 4.4.1.4) 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

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,

surface residue which is occluded by the coenzyme NADP+ (Fig.4)

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 (27). 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 (27). 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 (27).

Using ESR spectroscopy, we examined the availability of the free —SH groups of alliinase for chemical modification with the disulfide containing biradical **. RS-SR.** (27). The rate of the thiol–disulfide exchange reaction was monitored by ESR assay of the monoradical **•R—SH** released in this reaction, according to Eq. 2.

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 *p*-chloro mercury benzoate dramatically inhibited the modification kinetics (data not shown). Figure 6 shows the ESR spectrum of alliinase modified by the biradical (after 5 h of incubation followed by removal of the excess reagent by gel filtration) at 120 K. This spectrum is typical of a nitroxyl stable radical in a frozen solution. The degree of modification obtained by double integration of the ESR spectrum was 1.61 ± 0.15 per subunit of alliinase. To estimate the distance between two labeled cysteine residues, we used the empirical parameter *d*1/*d* (see Fig.6), which characterizes the dipole–dipole interaction between unpaired electrons of two nitroxyl groups, as proposed by Kokorin *et al* (28) and commonly used to estimate the distance between two radicals covalently bound to proteins (29, 30). In the absence of dipole–dipole interactions between the radicals, a value *d*1/*d* < 0.4 is expected. The value obtained for *d*1/*d* obtained in our experiment was 0.38, and the distance between the labeled cysteines Cys 220 and Cys 350 was estimated to be larger than 22 Å.

Alliinase has been crystallized and its three-dimensional structure solved (25-27). The enzyme is a homodimeric glycoprotein belonging to the fold-type I family of PLPdependent enzymes.

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 **. RS-SR.** (see Fig. 5). Distances between all the cysteines involved in disulfide bonds and the free thiols in the alliinase dimer range between 15 and 68 Å, and do not allow rearrangement of disulfide bonds in the native state.

**Figure 5.** Kinetics of the nitroxyl biradical modification of alliinase. Peak intensity of the ESR spectrum of the monoradical **(. R-SH)** component that was released into solution as a result of the thiol–disulfide exchange between SH groups of alliinase and the biradical **. RS-SR.** (see eq. 2) (from ref. 27)

**Figure 6.** ESR spectrum of the alliinase-biradical conjugate. The conjugate (12 μM) was measured in a PBS/glycerol (70/30) mixture at 120 K. ESR conditions: microwave power, 10 mW; modulation amplitude, 1.25 G;

Dimeric structure of alliinase is depicted in the illustration. Yellow and magenta indicate monomers A and B, respectively. Pyridoxal-5'-phosphate groups are shown as red spheres. The dimer is rotated 1800 around a vertical axis with respect to A. Free cysteines are shown as green spheres

**Figure 7.** Distribution of cysteines in a monomer of alliinase from garlic (*Allium sativum*). (from ref. 27)

### **4. Reversible modification of Thiol groups in proteins**

376 Nitroxides – Theory, Experiment and Applications

bonds in the native state.

of the monoradical **(.**

amplitude, 1.25 G;

exchange between SH groups of alliinase and the biradical **.**

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

5). Distances between all the cysteines involved in disulfide bonds and the free thiols in the alliinase dimer range between 15 and 68 Å, and do not allow rearrangement of disulfide

**Figure 5.** Kinetics of the nitroxyl biradical modification of alliinase. Peak intensity of the ESR spectrum

**Figure 6.** ESR spectrum of the alliinase-biradical conjugate. The conjugate (12 μM) was measured in a PBS/glycerol (70/30) mixture at 120 K. ESR conditions: microwave power, 10 mW; modulation

**R-SH)** component that was released into solution as a result of the thiol–disulfide

**RS-SR.**

(see eq. 2) (from ref. 27)

**RS-SR.** (see Fig.

each other (Fig. 8B), which might affect their chemical modification rates by **.**

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. (8) used the spin label MTSSL, for reversible chemical

modification of Cys 25 in papain. We made use of biradical **. RS-SR.** for the reversible chemical modification of NADPH-cytochrome P-450 reductase from rat liver (6,34), human hemoglobin (7), *Torpedo californica* acetylcholinesterase (*Tc*AChE) (35-37), TBADH (10,21) and allinase from garlic (27).

**Figure 8.** Free thiols of alliinase. (**A**) Relative locations of Cys220 and Cys350 (green) on the surface of the alliinase monomer. (**B**) Respective orientations of CysS220 and Cys350 relative to A. (from ref. 27)

#### **4.1. Acetylcholinesterase from** *Torpedo Californica* **(***Tc***AChE)**

Cys 231, a deeply buried residue in *Tc*AChE, was modified by biradical (eq.4) to yield a catalytically inactive species, even though it is not involved in the active site of the enzyme, which is a serine hydrolase (35-37).

$$\cdot \cdot \text{R} \cdot \text{s} \text{R} \cdot \text{s} \cdot \text{HS} \cdot \overbrace{\text{PR}}^{\cdot} \cdot \longleftrightarrow \cdot \text{R} \cdot \text{s} \cdot \overbrace{\text{PR}}^{\cdot} \cdot \cdot \text{R} \cdot \text{s} \text{H} \tag{4}$$

$$\cdot \text{RS} \cdot \text{S} \cdot \begin{pmatrix} \text{PR} \\ \text{S} \end{pmatrix} \cdot \text{HS} \cdot \text{G} \iff \cdot \text{RS} \cdot \text{SG} \cdot \text{HS} \cdot \begin{pmatrix} \text{PR} \\ \text{S} \end{pmatrix} \tag{5}$$

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, **MG**, conformation (38,39). In such cases, where the chemical modification induced unfolding of the protein, there was a concomitant complete disappearance of ellipticity in the near-UV of the CD spectrum (**λmin** = 280 nm), red shift of the maximum of intrinsic fluorescence spectrum (**333 nm 341 nm**) and enzyme inactivation. However, changes of the secondary structure were very modest (35). Chemical modification Cys 231 by organo-mercurials nitroxyl radical: (2,2,5,5-Tetramethyl-4-[2- (chloromercuri)phenyl]-3-imidazoline-1-oxy1 (**HgR.** ) and the natural thiosulfinate, allicin (see sheme 1), transforms *Tc*AChE to a quasi-native (**N\***) state (36,40). Note, that modification by **HgR.** and allicin was also reversible and modified AChE was demodified by treatment with reduced glutathione (eq 5). Demodification of the organomercurial (or allicin) modified enzyme with GSH shortly after modification leads to regeneration of the physicochemical characteristics of the native enzyme as well as to *Tc*AChE reactivation. The modified enzyme in **N\*** state is, however, metastable, and is converted spontaneously and irreversibly, at room temperature, with **t1/2** 100 min, to an **MG** state. Using the developed approach we were able to describe the conformational states of *Tc*AChE, transitions between these states (**N**- native, **U**-unfolded state), stimulation transition to MG state by biological membranes (41,42) as well as stabilization of **N** and **N\*** states by chemical and pharmacological chaperons (40,43)

$$\mathbf{N} \Leftrightarrow \mathbf{N^\*} \Leftrightarrow \mathbf{M} \mathbf{G} \Leftrightarrow \mathbf{U}$$

#### **Scheme 2.**

378 Nitroxides – Theory, Experiment and Applications

and allinase from garlic (27).

modification of Cys 25 in papain. We made use of biradical **.**

chemical modification of NADPH-cytochrome P-450 reductase from rat liver (6,34), human hemoglobin (7), *Torpedo californica* acetylcholinesterase (*Tc*AChE) (35-37), TBADH (10,21)

**Figure 8.** Free thiols of alliinase. (**A**) Relative locations of Cys220 and Cys350 (green) on the surface of the alliinase monomer. (**B**) Respective orientations of CysS220 and Cys350 relative to A. (from ref. 27)

Cys 231, a deeply buried residue in *Tc*AChE, was modified by biradical (eq.4) to yield a catalytically inactive species, even though it is not involved in the active site of the enzyme,

**4.1. Acetylcholinesterase from** *Torpedo Californica* **(***Tc***AChE)** 

which is a serine hydrolase (35-37).

**RS-SR.** for the reversible

**A**

**B**

(4)

(5)

### **4.2. TBADH and NADPH-cytochrome P-450 reductase**

Chemical modification of both TBADH and NADPH-cytochrome P-450 reductase by biradical **. RS-SR.** (6,10,21,34)) also led to their inactivation (see Fig. 9)

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 (***Allium sativum***)**

We have shown recently that modification of Cys 220 and Cys 350 of alliinase with **. RS-SR.** does not change its enzymatic activity (27). 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

**Figure 9.** Influence of NADP+ and DTT on effect of biradical **. RS-SR.** on TBADH activity. Enzyme was incubated with the biradical without (closed circles) and with (open circles) NADP+. DTT was introduced at 10 min (shown by arrow). (from ref. 21)

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).

**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.
