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Phenothiazine Derivatives as Enhancers of Peroxidase Dependent Chemiluminescence. M. M. Vdovenko, A. Kh. Vorobiev, and I. Yu. Sakharov1. Chemistry ...
ISSN 10681620, Russian Journal of Bioorganic Chemistry, 2013, Vol. 39, No. 2, pp. 176–180. © Pleiades Publishing, Ltd., 2013. Original Russian Text © M.M. Vdovenko, A.Kh. Vorob’ev, I.Yu. Sakharov, 2013, published in Bioorganicheskaya Khimiya, 2013, Vol. 39, No. 2, pp. 200–205.

Phenothiazine Derivatives as Enhancers of PeroxidaseDependent Chemiluminescence M. M. Vdovenko, A. Kh. Vorobiev, and I. Yu. Sakharov1 Chemistry Department, Moscow State University, Moscow, 119991 Russia Received May 10, 2012; in final form, May 21, 2012.

Abstract—Some Nalkyl phenothiazines with different ionic groups were studied as enhancers of chemilu minescence catalyzed by soybean peroxidase. It was shown that under experimental conditions, the com pounds with positively charged groups do not exhibit enhancing ability, while the addition of phenothiazines with negatively charged groups to a substrate mixture significantly increased the chemiluminescence inten sity. The relationship between the enhancing activity of phenothiazines and their capacity for enzymatic oxi dation by hydrogen peroxide was found. The enhancers discovered new opportunities for increasing the sen sitivity of determination of analytes by chemiluminescent enzyme immunoassay. Keywords: phenothiazine, peroxidase, chemiluminescence, enhancers DOI: 10.1134/S1068162013020155 1

INTRODUCTION At present, enzyme immunoassay (EIA) is one of the most common analytical methods used in various fields of medicine, agriculture, microbiology, food industries, and for monitoring the environment. It is due to the underlying high affinity and unique speci ficity of immunochemical reactions between antigens and antibodies, and also to the high sensitivity of detection of enzyme labels. In modern practice, a variety of EIA formats is applied, but at the last stage, regardless of the analysis for mat, the determination of the catalytic activity of the enzyme label is carried out. For this purpose the colori metric, fluorescent or chemiluminescent detection methods are used in EIA. The chemiluminescent detec tion method is the more sensitive of the above [1]. In the case when peroxidase is used as an enzyme label, luminol is used as a substrate for the chemilumi nescent registration of enzyme activity. It is known that the catalytic activity of peroxidase towards lumi nol is very low, so for increasing the recorded lumines cence intensity the introduction of additional com pounds called “enhancers” into a substrate mixture is required. In studies of the enhanced chemiluminescence reaction [2–4] a mechanism of simultaneous oxida

tion of luminol and an enhancer was postulated, which assumes that at the first stage a number of the enzy matic oxidation reactions of the enhancer take place in accordance with the “pingpong” mechanism: E + H2O2 ⇒ EI EII + SH ⇒ E + S•, where SH is the substrate enhancer; S• is the radical product of the oxidation of substrate enhancer; E, EI, and EII are the different forms of peroxidase. Further, a number of nonenzymatic reactions of the substrate enhancer and luminol proceed with the formation of 3aminophthalate in the electronically excited state. The transition of 3aminophthalate to the ground state is accompanied by emission of a light quantum (hν). S• + AH– ⇒ SH + A•–

Abbreviations: EIA, enzyme immunoassay; ECR, enhanced chemiluminescence reaction; HRP, horseradish peroxidase; SBP, soybean peroxidase; PTPS, sodium 3(phenothiazin10 yl)propane1sulfonate; PTP, sodium 3(phenothiazin10 yl)propionate; CPTP, sodium 3(2chlorophenothiazin10 yl)propionate. 1 Corresponding author: phone: +7 (495) 9393407; fax: +7 (495) 9395417; email: [email protected].

where AH– is the anionic form of luminol in alkaline medium, A•– is the radical product of luminol oxida tion, A is luminol diazaquinone, AO2H– is luminol peroxide, and 3AP is 3aminophthalate. A wide range of compounds, including derivatives of 6hydroxybenzothiazoles, substituted phenols,

EI + SH ⇒ EII + S•

•–

A•– + O2 ⇒ A + O 2 •–

A•– + O 2 + H+ ⇒ АO2H– 2 A•– ⇒ A + AH– A + HO2– ⇒ AO2H– AO2H– ⇒ 3AP + N2 + H+ + hν,

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naphthols, and anilines can accelerate the reaction of peroxidase oxidation of luminol [5–7]. At simulta neous oxidation, a substrate enhancer and luminol exhibit synergism, which is expressed in the nonaddi tivity of the observed signal and the signals for the indi vidual oxidation of substrates. Enhancement of chemiluminescence can reach several hundreds, and sometimes thousands, of times depending on the nature of the substrate enhancer, reagent concentra tions and reaction conditions [8, 9]. 4Iodophenol is the best known enhancer for the peroxidasedependent enhanced chemiluminescence reaction (ECR). The detection limit of horseradish peroxidase (HRP) in the ECR was 1.0 pM [10]. At the same time, it was recently demonstrated that one of the phenothiazine derivatives, namely sodium 3(phe nothiazin10yl)propane1sulfonate (PTPS) is a more promising enhancer of chemiluminescence cata lyzed by horseradish and soybean peroxidases [11, 12]. Thus, the detection limit in the case of PTPS was 0.03 pM [12]. S

In this paper, we attempted to estimate the pros pects of using other phenothiazine derivatives as the chemiluminescence enhancers and to establish the relationship between the chemical structure of phe nothiazines and their enhancing ability. RESULTS AND DISCUSSION The previously detected effect of enhanced chemi luminescence intensity in the ECR under the action of PTPS in the presence of plant peroxidases stimulated a comparative study of the phenothiazine derivatives containing different substituents as the potential enhancers of peroxidasedependent chemilumines cence. The following phenothiazine derivatives were selected for comparison: diprasine, chloracyzine, nonachlazine, ethacyzine, perphenazine, sodium 3 (phenothiazin10yl)propionate (PTP), sodium 3(2 chlorophenothiazin10yl)propionate (CPTP), and PTPS.

S

N

S

N

CH3

Cl

C2H5

N

O C CH2 CH2 N

C2H5 Chloracyzine

Nonachlazine

S

S O NH C O

N

O C CH2 CH2 N

N

Cl

C2H5 C2H5 N

C2H5

Ethacyzine

S N

Cl

N

O C CH2 CH2 N

N CH 3 H3C Diprasine

177

Cl

N

OH

Perphenazine

S

S

N

N

CH2 CH2 COONa

CH2 CH2 COONa SO3Na

Sodium 3(2chlorophenothiazin Sodium 3(phenothiazin10yl) Sodium 3(phenothiazin10yl) propionate propane1sulfonate 10yl)propionate (CPTP) (PTPS) (PTP)

All experiments on the evaluation of the enhancing effect of phenothiazines were carried out under iden tical conditions, namely under the conditions most RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY

favorable to the enzymatic cooxidation of luminol and PTPS catalyzed by SBP (50 mM Tris buffer, pH 8.3, containing 0.75 mM of luminol and 0.5 mM Vol. 39

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Influence of the phenothiazine derivatives on the value of the enhancing effect of chemiluminescence produced in their co oxidation with luminol in the presence of soybean peroxidase.* Experimental conditions: 50 mM Tris buffer (pH 8.3), [lu minol] = 0.75 mM, [H2O2] = 0.5 mM, [SBP] = 30 pM Concentration of phenothiazine derivative, mM

Enhancing effect

0.2

0.45 ± 0.06

Chloracyzine

0.2

0.345 ± 0.02

Nonachlazine

0.2

0.20 ± 0.02

Ethacyzine

0.2

0.19 ± 0.01

Perphenazine

0.2

5.52 ± 0.32

1.0

237 ± 5

CPTP

1.0

76 ± 5

PTPS

0.2

159 ± 9

1.0

240 ± 13

Phenothiazine derivatives With positively charged ionic groups Diprasine

With negatively charged ionic groups PTP

Note: *The enhancing effect was calculated as the ratio of the light intensities recorded in ECR in the presence and absence of phenothi azine.

Н2О2 [12]). As evident from the table, almost all of phenothiazine derivatives with positively charged ionic groups (diprasine, chloracyzine, nonachlazine, ethacyzine) introduced in the substrate mixture at a concentration of 0.2 mM due to their low water solu bility have a negative enhancing effect, i.e., they were not the luminescence enhancer but its, even very Absorbance 1.5

1.0 3

0.5

2 1 4

0

5

350

400

450

500

550 600 Wavelength, nm

Fig. 1. Change in the absorbance spectra of the reaction products in SBPcatalyzed oxidation of PTPS by hydrogen peroxide. Experimental conditions: 50 mM Tris buffer (pH 8.3), [PTPS] 1.0 mM, [H2O2] 0.5 mM, [SBP] 10 nM. Dif ferential spectra were recorded in (1) 3, (2) 17, (3) 27, (4) 51, and (5) 109 min after the start of the reaction.

weak, quenchers. From this group of substances, only perphenazine exhibited properties of the enhancer of peroxidasedependent chemiluminescence, but the magnitude of the enhancing effect of this substance was extremely low. On the other hand, the presence of phenothiazine derivatives with negatively charged ionic groups (PTPS, PTP, CPTP) in the substrate mixture increased the chemiluminescence intensity by a great extent. Thus, the addition of PTPS and PTP (1 mM) to the substrate mixture resulted in an almost 250fold increase in the chemiluminescence signal. In this case it was observed that the introduction of an additional substituent (chlorine) in the aromatic ring resulted in a lowering of the enhancing effect (table). So, under experimental conditions PTP and PTPS had the max imum enhancing effect, they contain in their structure the negatively charged carboxyl and sulfo group, respectively, that are attached to the phenothiazine core through the Npropyl group. The difference in the values of the enhancing effect that was found for the Nalkylated phenothiazine derivatives studied is likely to be due to their different oxidizability under the action of compound I (EI) and compound II (EII) of peroxidase. To test this hypoth esis, we studied the oxidation of phenothiazine deriv atives by hydrogen peroxide in the presence of SBP. First of all, the oxidation of PTPS was studied, for which purpose, during the reaction, we recorded the differential spectra of the substrate mixture in regular time intervals. Figure 1 shows that the spectrum of the products of the enzymatic oxidation of PTPS has two

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10 Gs

Fig. 2. ESR spectrum registered in SBPcatalyzed oxidation of PTPS by hydrogen peroxide. Experimental conditions, see the caption to Fig. 1.

characteristic peaks with maxima at 345 and 513 nm. It should be noted that if the curve with saturation corre sponded to the change in the peak intensity at 345 nm with time, the intensity of another peak first reached its maximum and then gradually decreased to zero. Based on the published data [2–4], we assumed that the first peak corresponds to the formation of PTPS sulfoxide, while the peak at 513 nm is associated with the formation of the PTPS radical cation [13]. It should be emphasized that in the absence of SBP we did not detect the appearance of characteristic peaks in the reaction medium. In addition to recording the changes in the UV and visible spectra, we also recorded the ESR signal during the reaction under investigation (Fig. 2). The shape of the signal corresponds to the shape of the ESR signal for a radical of chlorpromazine, one of the phenothia zine derivatives [14]. The coincidence of the kinetics of changes in the optical absorption at 513 nm and the integrated ESR signal (Fig. 3) confirmed the above assumption that the peak at 513 nm can be used as a parameter for recording the formation of the PTPS radical cation (Fig. 2). Thus, the spectral studies dem onstrated the capacity of PTPS for enzymatic oxida tion by hydrogen peroxide. Under the experimental conditions, the initial rate of formation of the radical cation of PTPS, which, in turn, should oxidize the luminol molecules in the ECR, was 0.075 ОU513/min. Under the conditions most favorable to the oxida tion of PTPS [12], the oxidation of other phenothiaz ine derivatives under investigation was carried out. This study showed that in the oxidation of phenothiaz ine derivatives with positively charged ionic groups the RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY

reaction products absorbing at 513 nm were not formed. In contrast to the phenothiazine derivatives with positively charged ionic groups, PTP and CPTP were subjected to the efficient oxidation in the pres ence of SBP. The rates of formation of the oxidation products which absorb at 513 nm were 0.091 and 0.015 ОU513/min for PTP and CPTP, respectively. It should be noted that the diammonium salt of 2,2'azinobis(3ethylbenzothiazoline6sulfonic acid), one of the most efficient peroxidase substrates [15, 16], also bears a negative charge. The presence of nega Signal, % 100 Absorbance at 345 nm Absorbance at 513 nm Integral ESR signal

80 60 40 20

0

20

10

30

40

50 60 Time, min

Fig. 3. The dependence of the signals registered spectro photometrically and by ESR on the time of the oxidation reaction of PTPS. Experimental conditions, see the cap tion to Fig. 1. Vol. 39

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tively charged groups, as in the case of PTP, CPTP, and PTPS, is likely to be essential for productive binding of the bulky substrates to the active site of peroxidases. Thus, in this study we revealed the relationship between the ability of phenothiazines to enzymatic oxidation with the formation of radical cations and their ability to enhance the luminescence intensity in the ECR.

ACKNOWLEDGMENTS The work was financially supported by the Russian Foundation for Basic Research (grants 110492005 NNS_a and 110492473MNTI_a). The authors would like to thank Dr. O.S. Sokolova (Biology Fac ulty of MSU, Russia) for providing the luminometer. REFERENCES

EXPERIMENTAL In this work we used peroxidase from soybean seed hulls (SBP, RZ 1.5) (Sigma, United States), hydrogen peroxide (KhimMed, Russia), luminol (Aldrich, United States), and sodium 3(phenothiazin10yl) propane1sulfonate that was synthesized as described in [11]. Diprasine, chloracyzine, nonachlazine, ethacyzine, perphenazine, PTP, and CPTP were kindly provided by Prof. S.A. Eremin (MSU). The concentration of hydrogen peroxide was determined spectrophotometrically (ε240 43.6 M–1 cm–1). All solu tions were prepared using distilled water. Enzymatic CoOxidation of Luminol and Phenothiazines by Hydrogen Peroxide in the Presence of Soybean Peroxidase Method A. For chemiluminescent tracking of enzymatic oxidation of luminol the experiment was performed as follows: 200 µL of 50 mM Tris buffer (pH 8.3) containing 0.5 mM hydrogen peroxide, 0.75 mM luminol, and 0.2 or 1.0 mM phenothiazine under investigation were added to the wells of a black opaque 96well plate for enzyme immunoassay (Maxi Sorp, NUNC, Denmark) at room temperature. Then the oxidation reaction was initiated by adding of 50 µL of SBP (30 pM). The chemiluminescence intensity and its change with time were measured in a Zenyth 1100&3100 luminometer for 96well plates (Anthos Labtec Instruments GmbH, Austria). Method B. For spectrophotometric tracking of enzymatic oxidation of phenothiazines, the experi ment was carried out as follows: 3.7 µL of SBP (2.7 × 10–6 M) was added to 1 mL of 50 mM Tris buffer (pH 8.3) containing 0.2 or 1.0 mM of phenothiazine derivatives and 0.5 mM H2O2. Kinetics of change in the differential spectra from 290 to 600 nm was mea sured using a UV2401 spectrophotometer (Shi madzu, Japan). Method C. When registering the ESR spectra (25°С, Varian E3 spectrometer (Palo Alto, United States) in the enzymatic oxidation of Nderivatives of phenothiazine, the experiment was performed as described in method B.

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