Voltammetric and Spectroscopic Studies on the Interaction of Pentoxifylline with Cysteine in the Presence and Absence of UV Irradiation

Z. Phys. Chem.219 (2005) 817–830 by Oldenbourg Wissenschaftsverlag, München

Voltammetric and Spectroscopic Studies on theInteraction of Pentoxifylline with Cysteine inthe Presence and Absence of UV Irradiation

By Ender Biçer∗ and Elif Çınar

Ondokuz Mayıs University, Faculty of Arts and Sciences, Department of Chemistry,55139 Kurupelit-Samsun, Turkey

(Received November 30, 2004; accepted in revised form March 23, 2005)

Voltammetry / Pentoxifylline / Cysteine / Interaction / UV Irradiation

The interaction of pentoxifylline (PTX), a methylxanthine derivative drug, with cysteineon the hanging mercury drop electrode studied by square-wave voltammetry, cyclicvoltammetry and UV-Vis spectroscopy at phosphate buffer (pH 7.4) in the presence andabsence of UV irradiation. The addition of PTX to cysteine solution results in the decreaseof peak currents of mercurous cysteine thiolate and the shift of its voltammetric reversiblepeak towards positive potentials. Electrochemical data indicated a 1: 1 molecular complexformation of PTX with cysteine by means of electrostatic interaction and intermolecularforces. For the long UV irradiation times (25–85 min), voltammogram of the cysteinesolution gave the reduction peaks of sulphonyl radical (−0.056 V), disulfidic anion(−0.684 V) and free cystine (−0.800 V) due to probably the formation of thiyl radical.With adding of PTX to the UV irradiated cysteine solution, a new peak at−0.292 V wasobserved. The peak at−0.292 V was also obtained for the cysteine and PTX mixtureunder UV irradiation, but the peaks of sulphonyl radical and disulphidic anion were noseen. As a result, this irreversible peak at−0.292 V may be assigned to the reductionof an electroactive species which is formed from PTX–cysteine complex under UVirradiation. PTX prevents the formation of thiyl radical and its further oxidation.

1. IntroductionPTX (Scheme 1) increases erythrocyte flexibility, reduces blood viscosityand improves microcirculatory flow and tissue perfusion [1, 2]. Numerousnovel clinical applications of PTX in dilated cardiomyopathy [3], nephropa-thy [4, 5] and cancer [6, 7] therapy have been proposed. PTX is also knownto possess anti-inflammatory properties [8] which are probably related to theirability to suppress oxygen radical production or scavenge reactive oxygen

* Corresponding author. E-mail: [email protected]

818 E. Biçer and E. Çınar

species [9]. Ability of PTX to scavenge hydroxyl radicals has been demon-strated earlier [10].

Bhat and Madyastha [11] reported that 8-oxopentoxifylline is significantlybetter hydroxyl and peroxyl radical scavenger than PTX.

The physiological importance of sulphydryl thiols is well established withthe levels of these compounds within biological fluids such as plasma andurine serving as valuable biomarkers in a number of clinical situations [12–15]. Cysteine (Scheme 1) is biologically important. This fact, combined withits electrochemical activity, has assigned its special interest among the aminoacids for voltammetric investigations [16].

Almost all drugs exert their pharmacologic effect by interactions with somekind of protein in the body and are eliminated either by combining with severaltransport systems or by drug-metabolizing enzymes [17].

No voltammetric references on the interaction of PTX with cysteine couldbe traced in the literature. It was therefore considered important to reportthe use of voltammetric techniques to monitor the interaction of PTX withcysteine in the presence and absence of UV irradiation at the physiologicalpH (7.40).

Scheme 1.

2. Experimental

2.1 Chemicals

PTX and cysteine were purchased from Sigma and Merck, respectively. Allchemicals were of analytical grade. The stock solutions were prepared in triplydistilled and deionized water and used immediately.

2.2 Apparatus

A three-electrode potentiostatic control system (EG&G PARC 303A SMDE)with a hanging mercury working electrode (HMDE), a Ag

∣∣AgCl

∣∣KClsat. ref-

Voltammetric and Spectroscopic Studies on the Interaction of Pentoxifylline. . . 819

erence electrode and a platinum auxiliary electrode has been used in all ex-periments. The potential scan was generated by means of a EG&G PAR 384BPolarographic Analyzer. The recording of current–potential curves was ob-tained by means of a Houston Instrument DMP-40 plotter connected to thepolarograph.

Philips Sunlamp (HP 3202) of 300 Watt was used as ultra-violet (UV) lightsource for UV-irradiation studies.

The electronic absorption spectra in the 400–200 nm range were recordedon Unicam V2–100 UV/Vis spectrophotometer, using quartz absorption cells(the length of the cells= 1 cm).

2.3 Procedure

Before voltammetric experiments, the solution within the electrochemical cellwas deareted by purging with pure nitrogen gas for 5 min, and during measure-ments a stream of nitrogen gas was passed over the solution. The voltammo-grams were obtained by using equilibrium time of 5 s; scan rate of 200 mV s−1

(500 mV s−1 for CV); scan increment of 2 mV; drop size, medium (unlessstated otherwise). In the absence of UV irradiation, the interaction betweencysteine and PTX was studied by using the amperometric titration. The re-duction peak current of mercurous cysteine thiolate (Hg2(RS)2) was followedwhen the PTX concentration was increased.

In the literature, the voltammetric studies of the interactions of DNA withmethylene blue (MB) [18] were studied by means of amperometric titrationmethod. The current titration equation on the binding of methylene blue (MB)with DNA was described as follows [18]:

1/Cp = K f(1− A)/(1− i/i0)− K f (1)

where,Cp is the concentration of DNA,K f is the apparent formation constant,i0 andi are the peak current without and with DNA.A is the proportional con-stant. The condition of using this equation is that a 1: 1 association complex isformed andCp is much larger than the total concentration of MB in solution. Inthe other word, if Eq. (1) corresponds well to the experimental data, this maysuggest that the complex of MB with DNA is a 1: 1 association complex. Inthis study, the interaction of cysteine with PTX was studied by using Eq. (1).All experiments were carried out at room temperature.

3. Results and discussion3.1 The interaction of pentoxifylline with cysteine in the absence of

UV irradiationThe square-wave voltammogram of 2.91×10−5 M PTX in phosphate bufferpH 7.40, in the absence of cysteine gave two peaks at−0.110 V and−1.524 V,


Fig. 1. The square-wave voltammogram of 2.91× 10−5 M PTX in phosphate bufferpH 7.40. Inset: The cyclic voltammogram (CV) of 2.91× 10−5 M PTX in phosphatebuffer pH 7.40. The experimental conditions: equilibrium time 5 s; scan rate 200 mV s−1

(500 mV s−1 for CV); scan increment, 2 mV; drop size, large. 1U, the reduction of Hg(I)-PTX complex; 2U, the reduction of carbonyl group on the extended aliphatic chain of thePTX molecule.

respectively (Fig. 1). These peaks have previously been attributed to the reduc-tion of Hg(I)-PTX complex and carbonyl group on the extended aliphatic chainof the molecule, respectively [19]. The formation of the first peak (−0.110 V)depends on the nature and/or pH of supporting electrolytes, and the concentra-tion of PTX.

The square-wave voltammogram of 2.91×10−5 M cysteine (RSH) in theabsence of UV irradiation exhibits two peaks at−0.124 V and−0.596 V inphosphate buffer (pH 7.40) (Fig. 2). These peaks are attributed to the reduc-tion of mercuric cysteine thiolate (Hg(RS)2) to mercurous cysteine thiolate(Hg2(RS)2) and the reduction of Hg2(RS)2 to metallic mercury and free thiolate(RS−) ions [20–22]. However, the reduction of Hg(RS)2 to Hg2(RS)2 in phos-


Fig. 2. The square-wave voltammogram of 2.91×10−5 M cysteine (RSH) in phosphatebuffer (pH 7.40).Inset: The cyclic voltammogram of 2.91×10−5 M cysteine (RSH) inphosphate buffer (pH 7.40). (Drop size, medium and other conditions as in Fig. 1). 1U,the reduction of mercuric cysteine thiolate (Hg(RS)2) to mercurous cysteine thiolate(Hg2(RS)2); 2U, the reduction of Hg2(RS)2 to metallic mercury and free thiolate (RS−)ions.

phate buffer (pH 7.40) was observed at the high cysteine concentrations ([RSH]≥ 1×10−5 M). The currents and potentials of these peaks also depend on theexperimental conditions and the concentration of cysteine.

Square-wave voltammetry were previously used to study the interaction be-tween the molecules [20, 23–25]. In order to study the interaction of PTX withcysteine, the peak of Hg2(RS)2 was selected. The increasing concentrations(from 3.90×10−4 M to 5.50×10−3 M) of PTX was added to the 4.98×10−6 Mcysteine solution. The peak current and peak potential of Hg2(RS)2 were foundto depend on the PTX concentration (Fig. 3). In Fig. 3, the peaks correspondingto the−0.110 V reduction of PTX and to the−0.124 V reduction of cysteine,respectively, are vanished as compared with Figs. 1 and 2. It can be owing to


Fig. 3. Effect of PTX on the potential and current of the voltammetric reduction ofHg2(RS)2. Square-wave voltammogram of solution of 4.98×10−6 M cysteine at HMDE inphosphate buffer (pH 7.40) in the absence (− −−) and presence (––––) of 2.14×10−3 MPTX. Scanning from positive to negative potentials at the rate of 200 mV s−1 (drop size,medium and other conditions as in Fig. 1). 1U, the reduction of Hg2(RS)2 to metallic mer-cury and free thiolate (RS−) ions; 2U, the reduction of carbonyl group on the extendedaliphatic chain of the PTX molecule.

the cysteine concentration and cysteine–PTX interaction. The peak current ofHg2(RS)2 decreased with increasing PTX concentration. The decrease of thepeak current corresponding to the reduction of Hg2(RS)2 can be ascribed toa decrease of the free cysteine concentration. Moreover, the decrease of thepeak current is caused by the fact that much of the cysteine exists as the moreslowly diffusing PTX–cysteine adduct. Taking account that the concentrationof PTX is higher than that of cysteine, most of the cysteine molecules shouldhave then bound to PTX and the peak current tends to be governed by diffusionof the PTX–cysteine complex.


Fig. 4. The plot of 1/[PTX] to 1/(1− i/i0).

In addition, the peak potential of Hg2(RS)2 shifted to more positive poten-tials owing to probably the interaction of PTX with cysteine (Fig. 3). The simi-lar results were observed for the interaction between folates and thiols [26, 27].The shift in the cathodic peak potential of Hg2(RS)2 is 130 mV. The reason ofpeak potential shifting to more positive value is that cysteine molecules expe-rienced a high electron density when they interacts with PTX, which made iteasy to accept electron. The above reported results show that the nature of theinteraction may be the cooperative binding due to the intermolecular forces.

According to the decrease of peak currents of Hg2(RS)2 with the concen-trations of PTX, the following equation was obtained: 1/[PTX] = 368.58/(1− i/i0)−250.15 with a linear correlation coefficient (r) of 0.998 (Fig. 4).This revealed that the interaction of PTX with cysteine was a 1: 1 associationcomplex and the formation constant (K f ) was 250.15 M−1 for cysteine-PTX, ascalculated from they-intercept [18].

The interaction between PTX and cysteine could be further confirmed bya spectroscopic experiment. The electronic spectra of PTX without and withcysteine are investigated. In the absence of cysteine, PTX gives a maximum ab-sorption peak at 268 nm (Fig. 5). On the other hand, cysteine solution withoutPTX exhibits a maximum absorption band at 229 nm (Fig. 5). When cysteinewas added to the PTX solution, a new absorption peak with different shape at274 nm was observed (Fig. 5). The absorption peak at 274 nm can indicate theinteraction between PTX and cysteine. But, the electronic transitions at 274 nmmay be also overlap with those of PTX.

3.2 The interaction of pentoxifylline with cysteine in the presence ofUV irradiation

The effect of UV light on the solution of cysteine was previously studied bycyclic voltammetry [28]. When the solution of 2×10−5 M cysteine was irradi-


Fig. 5. The electronic absorption spectra of solution of 2×10−4 M PTX in phosphatebuffer (pH 7.40) in the absence (a) and presence of (b) 4×10−5 M cysteine (––––). Theelectronic absorption spectrum of solution of 5×10−4 M cysteine in phosphate buffer(pH 7.40) (−−−).

ated for 15 min by UV light, a new irreversible peak at−0.792 V was appeared.The irreversible peak at−0.792 V was attributed to the free cystine [28].

In this study, 3.85×10−5 M cysteine solution was irradiated for long times(25–85 min) by UV light. For the long irradiation times, two new peaks wereobserved at−0.056 and−0.684 V, respectively while the reduction peak offree cystine was seen at−0.800 V (Fig. 6). With increasing the UV irradiationtime (25–85 min), the current of the peak at−0.684 V was increased and al-most fixed at 65 min and above (Fig. 7). The peak at−0.684 V has a weakadsorption characteristic and is reversible.

Cysteine thiyl radicals (RS·) are generated by one-electron oxidation pro-cess of cysteine or a homolytical cleavage of the S–H bond (Eqs. 2 and 3).

RSH→ RS·+ H+ +e− (2)

RSH→ RS·+ H· (3)

One-electron oxidation can be facilitated by a suitable oxidant. The energysupplied by a UV lamp is sufficiently large for the homolytical cleavage [29].


Fig. 6. Cyclic voltammogram of 3.85×10−5 M cysteine solution, exposed to UV irradi-ation of 65 min in phosphate buffer (pH 7.40), in the absence (− −−) and presence of(––––) 1.89×10−5 M PTX (drop size, medium and other conditions as in Fig. 1). 1U, thereduction of RSO2· radical; 2U, the reduction of mercuric cysteine thiolate (Hg(RS)2) tomercurous cysteine thiolate (Hg2(RS)2); 3U, the reduction of adduct of cysteine with PTXin the presence of UV irradiation; 4U, the reduction of Hg2(RS)2 to metallic mercuryand free thiolate (RS−) ions; 5U, the redox process of the disulfidic anion S2

2−; 6U, freecystine.

The further oxidation of thiyl radical may be carried out as following equa-tions [30]:

RS·+ 2H2O → RSO2·+ 4H+ + 4e− (4)

RS·+ 3H2O → RSO3− + 6H+ + 5e− . (5)

RSO3− is electroinactive but RSO2· radicals are reduced in the region of 0.6

to 0.0 V (vs. NHE) at platinum electrode in aqueous solutions [30]. With in-creasing UV irradiation time (25–85 min), the current of peak at−0.056 V alsoincreases and gave a maximum at 65-min (Fig. 7). This peak can be assigned tothe reduction of RSO2· radical.


Fig. 7. The effect of irradiation time on the peak current of the disulfidic anion S22− at

−0.684 V ( ) and of the reduction of RSO2· radical at−0.056 V ( ) in the solution of3.85×10−5 M cysteine.

Thiyl radicals can undergo dimerization to yield disulfides which terminatethe reaction [31]:

2RS· → RSSR. (6)

From all of the identified decomposition products of cystine (RSSR) pho-tolysis [32], the only species which is polarographically reducible and can betransformed into S-sulfocysteine (RSSO3

−) is the disulfidic anion S22− [33].In the previous paper [34], square-wave adsorptive stripping voltammetricbehaviour of fresh and aged cystine solutions at physiological pH (7.4) wasreported. The reduction of the disulfidic anion S2

2−, the decomposition prod-uct of cystine at physiological pH (7.4), was seen at−0.68 V (S2

2− +2e− +2H2O� 2SH− + 2OH−) [34] for aged cystine solution (a few hours) in thepresence of sunlight and air. In the present study, the peak (−0.684 V) may beassigned to the redox process of the disulfidic anion S2

2−. Probably, UV-lightaccelerate the formation of thiyl radical for the observation of cystine and thencystine decomposition.

When 1.89×10−5 M PTX was added to the irradiated (65 min) 3.85×10−5 M cysteine solution, the peak currents of Hg(RS)2, Hg2(RS)2, disulfidicanion and RSO2· radical decreased and a new irreversible peak at−0.292 Vwas observed (Fig. 6). It is expected that the current of peak at−0.292 Vshould increase gradually with increasing PTX concentration, because moreand more electroactive adducts will beformed with increasing of PTX. But,it was observed that the current of peak at−0.292 V decreases by increasingPTX concentration (Fig. 8). This may be the desorption of electroactive prod-uct at−0.292 V. The peak at−0.292 V can be ascribed to the reduction of anelectroactive species that is formed from the adduct of PTX with cysteine orthe disulfidic anion S22− in the presence of UV irradiation. On the other hand,


Fig. 8. The effect of PTX concentration on the current of the peak formed at−0.292 V for1.89×10−5 M PTX and 3.85×10−5 M cysteine mixture at phosphate buffer (pH 7.40) inthe presence of UV irradiation.

Fig. 9. Absorbancevs. UV irradiation time plot atλ = 274 nm for 2×10−4 M PTX and4×10−5 M cysteine mixture in phosphate buffer (pH 7.40).

PTX can behave as a radical scavenger. So, PTX scavenges thiyl or RSO2· rad-ical which formed due to the UV irradiation. Moreover, it was observed thatthe peak (6 U) corresponding to the reduction of free cystine disappeared afteraddition of PTX (Fig. 6). This case may be because of the scavenging of thiylradical (RS·).

To clarify the interaction of PTX with cysteine, the solution of cysteinewhich is including PTX was irradiated by UV light. The effect of UV-irradiation time on the absorbance seen at 274 nm for the PTX and cysteinemixture was also studied (Fig. 9). Fig. 9 showed that the absorbance at 274 nm


Fig. 10. Cyclic voltammogram of 7.71×10−4 M PTX and 9.17×10−6 M cysteine mixtureat phosphate buffer (pH 7.40) in the absence (−−−) and presence (––––) of UV irradi-ation of 5 min (drop size, medium and other conditions as in Fig. 1). 1U, the reductionof mercuric cysteine thiolate (Hg(RS)2) to mercurous cysteine thiolate (Hg2(RS)2); 2U,the reduction of adduct of cysteine with PTX in the presence of UV irradiation; 3U, thereduction of Hg2(RS)2 to metallic mercury and free thiolate (RS−) ions.

firstly reached a maximum value and, then decreased and fixed by increasingUV-irradiation times. According to this result, it can be said that the formedmolecular complex between PTX and cysteine is not very stable.

In the presence of UV irradiation, a new irreversible peak at−0.292 Vwas appeared on the cyclic voltammogram while this peak was no observedfor the PTX and cysteine mixture in theabsence of UV irradiation (Fig. 10).With increasing UV-irradiation time, the current of peak at−0.292 V increasesand then reaches a plato region (Fig. 11). Also, under the UV-irradiation thecyclic voltammogram of cysteine and PTX mixture does not exhibit the re-duction of RSO2· radical at−0.056 V (Fig. 10). Therefore, it can be said thatPTX prevents the formation of thiyl radical from cysteine and its further oxi-


Fig. 11. The effect of UV irradiation time on the current of the peak at−0.292 V for7.71×10−4 M PTX and 9.17×10−6 M cysteine mixture at phosphate buffer (pH 7.40).

dation. Finally, the peak at−0.292 V can be inferred from the reduction of anelectroactive species which is formed from PTX–cysteine complex under UVirradiation.

4. Conclusion

In the present paper, the electrochemical results indicated that the interactionof PTX with RSH occurs. Moreover, the binding model of cysteine to PTXis based on electrostatic binding and 1: 1 molecular complex formation. Ac-cording to the obtained results, it can besuggested that the interaction betweenPTX and cysteine plays an important role in the prevention of the formation orthe efficient removal of dangerous thiyl radicals. The investigation of the in-teraction of PTX with cysteine is alsouseful in understanding the mechanismof the interaction of PTX in the living body. On the other hand, voltammetrictechniques can potentially be used to characterize the interaction of any elec-troactive species with the drugs. Also,the voltammetric method for calculatingthe formation constant is more sensitive, easier and simpler.

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Documents

Voltammetric and Spectroscopic Studies on the Interaction of Pentoxifylline with Cysteine in the Presence and Absence of UV Irradiation