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Effect of albumin on the kinetics of ascorbate oxidation
Biochimica et Biophysica Acta 1526 (2001) 53^60
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E¡ect of albumin on the kinetics of ascorbate oxidation Evgenia Lozinsky a
a;
*, Artem Novoselsky a , Alexander I. Shames b , Oshra Saphier a , Gertz I. Likhtenshtein a , Dan Meyerstein a;c
Department of Chemistry, Ben-Gurion University of the Negev, 84105 Beer-Sheva, Israel b Department of Physics, Ben-Gurion University of the Negev, Beer-Sheva, Israel c College of Judea and Samaria, Ariel, Israel
Received 27 October 2000; received in revised form 23 January 2001; accepted 25 January 2001
Abstract The fluorescence intensity of the fluorophore in dansyl piperidine-nitroxide is intramolecularly quenched by the nitroxyl fragment. Therefore, the oxidation of ascorbic acid by the fluorophore-nitroxide (FN) probe can be monitored by two independent methods: steadystate fluorescence and electron paramagnetic resonance. Bovine serum albumin (BSA) affects the rate of this reaction. The influence of BSA on the rate is attributed to the adsorption of both ascorbate and the probe to BSA. Adsorption of ascorbate to BSA is confirmed by NMR relaxation experiments. The spatial distribution of the molecules on the BSA surface changes the availability of ascorbate and FN to each other. The results also point out that, in the presence of BSA, the autoxidation of ascorbate is significantly slowed down. The effect is studied at different pH values and explained in terms of the electrostatic interaction between the ascorbate anion and the BSA molecule. ß 2001 Elsevier Science B.V. All rights reserved. Keywords : Fluorophore-nitroxide ; Ascorbate oxidation; Bovine serum albumin
1. Introduction Recently nitroxyl spin labels have been extensively used as redox probes to measure the antioxidant status of biological liquids [1^3]. The only natural antioxidant which has been found to reduce nitroxides is ascorbate, AH3 [4]. Novel improved spin labels with extended properties and possibilities have been recently introduced [5^8]. These compounds contain a £uorophore (F) and a nitroxide (N). An example of this type is the dansyl piperidine-nitroxide used in the present study:
The covalently linked nitroxide radical quenches the £uorescence intensity of the £uorophore; therefore, the electron paramagnetic resonance (EPR) signal decay due
* Corresponding author. Fax: +972-7-647-2903; E-mail : [email protected]
to its reaction with AH3 is accompanied by the simultaneous enhancement of the £uorescence intensity. Thus two independent spectroscopic methods might be used to follow the reaction : steady-state £uorescence and EPR. Naturally, the increase of the £uorescence intensity and the decay of the EPR signal occur with the same rate. The reaction between the FN probe and excess ascorbate obeys a pseudo-¢rst-order rate law. The dependence of the observed rate on ascorbate concentration may be used as a calibration line for the determination of unknown concentrations of AH3 [5]. Thus, the FN probe might be used as a convenient tool for the estimation of the ascorbate content of di¡erent samples. However, attempts to use this calibration line for the measurements in more complicated solutions failed, i.e. the calibration line obtained in bu¡er solutions cannot be applied directly to many samples from living organisms, e.g. plasma and blood. It seemed plausible that this e¡ect is due to the interaction of ascorbate and/or the FN probe with proteins. Since albumin accounts for approx. 60% of the total protein in blood serum with a concentration of about 0.6 mM (40 g/l) [9], its solutions can serve as a model of mammalian plasma. The choice of albumin as a model was also based on the observation that it participates in the transport, distribution and metabolism of many endogenous and exogenous spe-
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cies. This transport function stems from the very high a¤nity of many organic and inorganic compounds (e.g. fatty acids, amino acids, steroids, metal ions, numerous drugs and dyes) to albumin. Albumin has many, and various binding sites which enable binding of many molecules [10,11]. It was decided therefore to study the in£uence of BSA on the kinetics of the reaction between dansyl piperidinenitroxide and ascorbate (AH3 ). The results indeed point out that both AH3 and FN are adsorbed to BSA and that this adsorption a¡ects the kinetics of the reaction between AH3 and FN. 2. Materials and methods 2.1. Materials Ascorbic acid, amino-TEMPO (4-amino-2,2,6,6-tetramethylpiperidinooxy nitroxide), TEMPOL (4-hydroxy2,2,6,6-tetramethylpiperidinooxy nitroxide) and BSA (fatty acid free) powders were purchased from Sigma. The FN probe was synthesized according to the procedure described in the literature [12], i.e. by mixing the commercial amino-TEMPO with dansyl chloride in pyridine. Stock solutions (0.01 M) of the FN probe were prepared in DMSO :water (3:7) and diluted prior to use. Aqueous solutions of BSA, TEMPOL and ascorbic acid (1 mM and 10 mM) served as stock solutions. The solution of ascorbic acid was kept under N2 before use. 2.1.1. EPR EPR spectra were recorded using a Bruker EMX-220 digital X-band spectrometer equipped with a Bruker ER 4241VT temperature control system. Solutions for analysis were prepared from stock solutions by dilution in PBS. Liquid samples were drawn into 1 mm i.d. glass Pasteur pipettes sealed at the bottom. Spectra at T = 120 K were recorded with 9.40 GHz microwave frequency, 0.20 mW non-saturating microwave power, 100 KHz ¢eld modulation of 0.05 mT amplitude. All spectra at T = 297 K were recorded with the following parameters: 9.40 GHz microwave frequency, 20.12 mW non-saturating microwave power, 100 kHz ¢eld modulation of 0.1 mT amplitude. Kinetics of the decay of the EPR signal of non-immobilized probe molecules were measured by ¢xing the magnetic ¢eld at the top peak position of the high ¢eld hyper¢ne line (M = +1) and recording in the Time Scan mode. Kinetics of the decay of the EPR signal of immobilized probe molecules were measured in the same manner by ¢xing the magnetic ¢eld at the position of the corresponding low ¢eld hyper¢ne line using the Time Scan mode and modulation amplitude 0.5 mT. The total error in the signal amplitude determination during the kinetic measurements was about þ 15%. The time delay between the initial mixing and the start of the scans in every experiment did not
exceed 80 s. Both EPR spectra and kinetics were processed using the Bruker WIN-EPR and Microcalc Origin software. 2.1.2. Steady-state £uorescence Fluorescence emission spectra and kinetics of the steady-state £uorescence increase were recorded using an ISS-GREG90-MM multifrequency phase modulation spectro£uorimeter SLM 4800 with a 450 W xenon lamp. Liquid sample solutions were prepared directly in the cuvette from stock solutions by dilution in PBS. Spectra were obtained using the following parameters : excitation wavelength 360 nm, scale 60%. All other spectral parameters are given in the legends of the ¢gures. The total error in signal amplitude determination during the kinetic measurements was about þ 10%. The data from £uorescence measurements were processed with KaleidaGraph software. 2.1.3. Nuclear magnetic resonance (NMR) 1 H NMR spectra (5 mm sample tubes, 298 K) and the measurement of 1 H T2 relaxation times were performed at 500.1 MHz on a Bruker DMX-500 Fourier transform spectrometer. 2 H2 O was used as an internal lock, and the residual H2 O solvent was used as an internal reference (NH = 4.8). Standard Bruker microprograms were utilized for the CPMG (Carr-Purcell-Meiboom-Gill) technique [13]. The error of the T2 measurement was smaller than þ 10%. 3. Results and discussion In order to check whether the FN probe is adsorbed to BSA, its EPR and steady-state £uorescence spectra in PBS and in BSA containing solutions were measured. Both methods demonstrate that the FN probe is bound to the albumin matrix (Fig. 1A,B). The EPR spectrum shows signi¢cant changes in the line shape attributing to the FN molecules incorporated into BSA (see additional signals marked by arrows in Fig. 1A). The shift of the £uorescence maximum to the blue points out that the binding occurs via the £uorophore fragment. Other EPR experiments were done to elucidate whether the FN probe is also bound to BSA through its nitroxide fragment. Parallel hyper¢ne splitting 2A0zz obtained from the spectrum of frozen solution is known to be a measure of the local polarity. We measured the 2A0zz values from the EPR spectra of FN in frozen bu¡er solution (at T = 120 K) in the presence and absence of BSA and found them to be the same: 2A0zz = 7.4 mT. It may be concluded that the nitroxide fragment has no a¤nity to the protein matrix and therefore most probably at room temperature it freely rotates in the aqueous phase. Such a conclusion is corroborated by the observation of EPR spectra of TEMPOL, which is an analogue of the radical fragment of FN. Nei-
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Fig. 1. Binding of FN (0.1 mM) to BSA. (A) EPR spectra of FN in PBS (999) and BSA (0.5 mM) (9 9 9) solution. Arrows show the signals of the immobilized probe. The spectrum in BSA solution was recorded at a 4-fold receiver gain. (B) Fluorescence emission spectra of free FN (0.1 mM) in PBS (999) and in solutions containing various concentrations of BSA (9, 0.01 mM; - - -, 0.05 mM; - 999 -, 0.1 mM). Emission and excitation slits 16 nm, scan rate 10 nm/s. All measurements were conducted at pH 7.4.
ther room nor low temperature spectra are a¡ected by the addition of BSA to its solutions, i.e. TEMPOL is not adsorbed to BSA. Like most small negatively charged organic molecules, ascorbate has a very high a¤nity to BSA, as demonstrated by a dialysis experiment [14]. In order to obtain a better understanding of the type of interaction between ascorbate and BSA, the NMR transverse relaxation times T2 of the
ascorbate protons in 2 H2 O and in solutions containing BSA were measured. The results for the methyl-ol proton (marked by `b' in Fig. 2) are summarized in Table 1. The decay of the transverse magnetization in pure 2 H2 O ¢ts a single exponent, with T2 = 765 ms. However, in the presence of BSA, the decay can be ¢tted only by the sum of two exponents. Binding of the ascorbate molecule to the massive protein causes a drastic decrease in the T2 of the
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Fig. 2. NMR spectrum of vitamin C (5 mM) after overnight incubation. Solvent 2 H2 O, pD 3.8.
ascorbate protons. The values of T2 for the two components (bound and free) are practically independent of the BSA content, while their ratio varies. The results point out that vitamin C is not bound tightly to BSA since the NMR lines of protons of the bound molecules are still observed. Moreover, all the ascorbate protons observed in the NMR spectrum relax with di¡erent rates, indicating that the mobility of the methine and methylene groups of the bound molecules di¡ers (Table 2). In order to estimate the contribution of BSA to the solution viscosity, the same experiment was performed with acetone, a small organic molecule that does not bind to albumin and possesses a very long T2 . The decrease in T2 of the acetone protons was found to be less than 20% from the initial value at 0.25 mM BSA concentration, while for ascorbate protons the decrease in T2 is 8.5^60-fold for the bound molecules at the same BSA content. These results show that the shortening of the transverse relaxation time is caused mainly by the binding of the ascorbate to albumin. Moreover, in the case of acetone the decay of the transverse magnetization always ¢ts a single exponential and is practically independent of the albumin content. The experimental results (Table 1) demonstrate that about 100 molecules of ascorbate can be adsorbed to one molecule BSA. This conclusion di¡ers from that reported in the literature Table 1 Percentage of bound and free components of ascorbate (5 mM) in BSA containing solutions and in the absence of BSA [BSA] (mM) Component 1 (765 ms) (%) Component 2 (90 ms) (%) pD 3.8.
0
0.001
0.01
0.05
0.025
100 0
87 13
85 15
68 32
10 90
where it was suggested that ten is the maximal number of the bound ascorbate molecules for one molecule of BSA [14]. In the presence of BSA, the transverse relaxation times of the ascorbate protons are pH-dependent (Fig. 3). The results clearly point out that T2 is shortest at pH 5.0 and becomes longer at both pH 3.8 and 7.0. As the ¢rst pKa of ascorbate is AH2 H2 O1AH3 H3 O pK a 3:8
1
and as AH3 is expected to be bound stronger to BSA, i.e. K1
AH3 BSAAH3 ÿ BSA
2
K2
AH2 BSAAH2 ÿ BSA
3
K1 s K2 , one expects an increase in the binding when the pH is raised from 3.8 to 5.0. The observed decrease at pH 7.0 is attributed to the isoelectric point of albumin at pH 5.2 [15]. The binding of AH3 to the negatively charged BSA at pH 7.0, although naturally positively charged substituents still are present on BSA at this pH, is clearly weaker. The reaction between the FN probe and AH3 was investigated by two independent techniques: EPR and Table 2 Values of T2 for free (component 1) and bound (component 2) ascorbate protons Proton (ppm) Component 1 (ms) Component 2 (ms)
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a(4.84)
b(3.97)
c(3.66)
820 60
765 90
245 4
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Fig. 3. Decay of transverse magnetization of the ascorbate proton (marked as `b' in Fig. 2) at di¡erent pD values (E, 3.8; tion of ascorbic acid and BSA 5 mM and 0.05 mM, respectively. Solvent 2 H2 O.
steady-state £uorescence. In the presence of excess ascorbate, the kinetic curves obey a ¢rst-order rate law. The observed rates, kapp , are proportional to [AH2 ] (Fig. 4). Addition of up to 1 mM BSA to the solution accelerates the observed rate. The e¡ect of BSA on the rate of the reaction has a clear concentration-dependent character (Fig. 4). From the slopes of the lines in Fig. 4 the rate constants for the reaction of ascorbate with FN (remembering that two FN molecules are reduced by each ascor-
57
a,
5.0; O, 7.0). Concentra-
bate and assuming that the ¢rst reaction is the rate determining step) are calculated. In the presence of 0.1 mM and 0.8 mM BSA, the rate constants of the reaction were found to be 25, and 38 M31 s31 respectively, while k = 13 M31 s31 in the absence of BSA. At low concentrations of BSA, the e¡ect of albumin on the rate constant is insigni¢cant. At higher concentrations the reaction is accelerated; however, not all FN molecules are reduced though [AH3 ] s [FN]. At 1 mM and higher concentra-
Fig. 4. Dependence of the observed rate of FN (0.1 mM) reduction on ascorbate concentration at di¡erent concentrations of BSA (a, no BSA; mM ; O, 0.8 mM). EPR measurements. PBS (pH 7.4); T = 297 K.
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0.1
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Fig. 5. E¡ect of pH on the rate of the reaction of FN (0.1 mM) with ascorbate in the presence of 0.1 mM BSA. O, pH 4.0;
tions of BSA, the EPR signal decay or/and the £uorescence enhancement processes are too fast to be measured in the CW experiment. A large portion of the FN which, according to its EPR spectrum, is all bound to BSA, is long lived in the time scale of the experiments: t1=2 s 10 min. Dilution of this solution results in the reappearance of the measurable kinetics: the EPR signal starts to decay and the £uorescence intensity grows. It should be noted that the reaction of TEMPOL with ascorbate, as followed by the EPR technique, is not a¡ected by the presence of BSA. The high resolution of the EPR spectrum of FN in BSA containing solutions enables the recording of the decay of the immobilized component as well as the free one. In all cases, the immobilized and the free components decay with the same rate constant, though as stated above a part of the immobilized FN remains at the end of the reaction. This result points out that in the system three components of the FN probe are present: (1) not adsorbed, free FN in the solution ; (2) loosely bound FN to BSA; this fraction is in fast equilibrium with the free FN, and therefore their rate of reaction with ascorbate is equal ; (3) tightly bound FN, which reacts slowly with the ascorbate either inherently or due to a low exchange rate with the other types of the probe. The observations can be explained by the spatial distribution of ascorbate and FN, which are adsorbed to BSA. When BSA is present at low concentrations, molecules of FN and ascorbate bind to the same BSA molecule. The sites of the primary binding are close; therefore, the probability of collision increases and the reaction proceeds faster. At high concentrations of BSA, the number of
a,
pH 5.0;
E,
pH 7.0.
available sites on BSA for FN and AH3 is large. A part of the FN and AH3 molecules are located at remote sites of BSA or are even bound to di¡erent BSA molecules. Thus they are separated from each other in space and the reaction is slowed down. This explanation is supported by the observation that the percentage of FN consumed in the reaction decreases with the increase in the concentration of BSA. The EPR spectrum of the residual amount of FN is observed after the reaction is over, though excess of ascorbate is present in the solutions. At 1 mM BSA, almost 100% of the FN remains bound to albumin and the reaction of these FN molecules with ascorbate is not observable in the time scale of the measurement (20 min). It was observed that even at relatively low concentrations of BSA (0.01^0.1 mM), a weak EPR signal of bound residual FN exists. At 0.1 mM BSA it achieves about 30% of the FN added. The estimation of the exact content of the rest of the FN at lower BSA concentrations is di¤cult because of the weak EPR signal and, correspondingly, low signal/ noise ratio. As the e¡ect of BSA on the rate of the reaction between FN and ascorbate is attributed to their binding to BSA, this e¡ect is expected to be pH-dependent. Indeed, such a dependence is observed ; the observed rate constants are 14, 87 and 42 M31 s31 at pH 4.0, 5.0, and 7.0, respectively (Fig. 5). The results are consistent with the pH dependence of the ascorbate binding to BSA (Fig. 3). The e¡ect of pH on the rate of reaction of FN with ascorbate in the absence of BSA was measured in bu¡ered solutions; the observed rate constants are 11, 19 and 14 M31 s31 at pH 4.0, 5.0 and 7.0 respectively. The small decrease in the rate between pH 5.0 and 7.0 is attributed to the pKa of the amino group of the probe.
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Fig. 6. Binding of FN (0.1 mM) to BSA. The binding ratio was calculated using the formula Ratio = (133DIN3 /DIN)U100%, where DIN is the normalized double integral of the whole EPR signal and DIN3 is the normalized double integral of the high-¢eld non-immobilized component, which has only a negligible overlap with the signal of the bound FN. O, pH 4.0; a, pH 5.0; E, pH 7.0; U, pH 7.4.
These results clearly demonstrate that the major factor a¡ecting the rate of the reaction between FN and vitamin C in the presence of BSA is the binding of both AH3 and FN to BSA. The e¡ect of pH on the binding of FN to BSA was also measured as another plausible contribution to the kinetics of ascorbate oxidation by FN. The results presented in Fig. 6 indicate that this binding increases from pH 4.0 to 5.0, probably due to the pKa of the amino
group of the FN. Above pH 5.0 only minor e¡ects are observed. The reaction of ascorbate with FN can also be used to follow the e¡ect of BSA on autoxidation of ascorbate. Ascorbate is oxidized by dioxygen in homogeneous neutral aqueous solutions [15]. In the presence of several proteins, including BSA, the rate of this process is signi¢cantly slowed down. Indeed, the EPR signal of FN does not
Fig. 7. Protective e¡ect of BSA on the kinetics of ascorbate autoxidation. Observed rate of FN (0.1 mM) reduction in a freshly prepared solution (a) and in a solution incubated overnight containing ascorbate and BSA (F) vs. BSA concentration. Concentration of ascorbate 0.1 mM. pH 7.4; T = 297 K.
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disappear when the probe is added to an ascorbate solution which was kept overnight at free access to dioxygen. Further addition of FN to an aerated solution of ascorbate and BSA which was kept for 14 h results in an EPR signal decay, pointing out that the ascorbate was preserved against autoxidation (Fig. 7). The NMR spectra of ascorbate, kept overnight in the presence and absence of albumin, also point out that BSA protects AH3 from autoxidation. In the absence of BSA the NMR of dehydroascorbate is clearly observed (Fig. 2). When the solution of AH3 contains BSA the peaks of the dehydroascorbate do not appear. 4. Concluding remarks The use of the FN probe, and probably other probes, for the determination of the ascorbate concentration in biological systems which contain proteins is hindered due to the protein concentration-dependent interactions between the proteins and/or the ascorbate and the probe. The addition of BSA to a mixture of FN and ascorbate a¡ects the kinetics of the reaction in a concentration-dependent fashion. Two, in principle opposing, e¡ects are observed: (1) the rate of the reaction is accelerated with increasing BSA concentration; (2) however, with the increase in BSA concentration a large part of the probe becomes `unreactive', though excess ascorbate is present in the solution. These e¡ects are attributed to the adsorption of both the probe and the ascorbate to the protein thus (a) bringing them closer to each other at low protein concentrations and (b) separating them spatially at high BSA concentrations. The binding of ascorbate to BSA is shown to be pHdependent due to two reasons: (1) the pKa of ascorbate; (2) the total charge of the BSA molecule. The electrostatic interaction between ascorbate and the protein decreases considerably the rate of its autoxidation.
References [1] N. Kocherginsky, H.M. Swartz, Nitroxide Spin Labels, CRC Press, Boca Raton, FL, 1995, pp. 114^172. [2] G. Likhtenshtein Spin Labeling Methods in Molecular Biology, Wiley-Interscience Press, 1982. [3] P.A. Motchnik, B. Frei, B.N. Ames, Measurement of antioxidants in human blood plasma, Methods Enzymol. 234 (1994) 269^279. [4] B. Frei, L. England, B.N. Ames, Ascorbate is an outstanding antioxidant in human blood plasma, Proc. Natl. Acad. Sci. USA 86 (1989) 6377^6381. [5] E.M. Lozinsky, V. Martin, T. Berezina, A. Shames, A. Weis, G.I. Likhtenshtein, J. Biophys. Biochem. Methods 38 (1999) 29^42. [6] S. Pou, Y.I. Huang, A. Bhan, V.S. Bhadti, R.S. Hosmane, S.Y. Wu, G.L. Cao, G.M. Rosen, A £uorophore-containing nitroxide as a probe to detect superoxide and hydroxyl radical generated by stimulated neutrophils, Anal. Biochem. 212 (1993) 85^90. [7] S.E. Herbelin, N.V. Blough, Intramolecular quenching of excited singlet states in a series of £uorescamine-derivatized nitroxides, J. Phys. Chem. B 102 (1998) 8170^8176. [8] S.A. Green, D.J. Simpson, G. Zhou, P.S. Ho, N.V. Blough, Intramolecular quenching of excited singlet by stable nitroxyl radicals, J. Am. Chem. Soc. 112 (1990) 7337^7346. [9] B. Halliwell, Albumin ^ an important extracellular antioxidant?, Med. Pharmacol. 37 (1988) 569^571. [10] X.M. He, D.C. Carter, Atomic structure and chemistry of human serum albumin, Nature 358 (1992) 209^214. [11] D.C. Carter, X.-M. He, S.H. Munson, P.D. Twigg, K.M. Gernert, M.B. Broom, T.Y. Miller, Three-dimensional structure of human serum albumin, Science 4 (1989) 1195^1198. [12] G.I. Likhtenstein, V.R. Bogatyrenko, A.V. Kulikov, K. Hideg, H.O. Hankovsky, N.V. Lukoianov, A.I. Kotelnikov, B.S. Tanaschelchuk, Study of superslow motion in solid solutions by the method of physical probes, Dokl. Akad. Nauk SSSR 253 (1980) 481^484. [13] T.C. Farrar, E.D. Becker, Pulse and Fourier Transform NMR. Introduction to Theory and Methods, Academic Press, New York, 1971. [14] V.C. Okore, E¡ect of ascorbic acid on the binding of cloxacillin sodium to bovine serum albumin, Arzneim.-Forsch. Drug Res. 44 (1994) 671^673. [15] Y. Wen, P. Dubin, Potentiometric studies of the interaction of bovine serum albumin and poly(dimethyldiallylammonium chloride), Macromolecules 30 (1997) 7856^7861.
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E¡ect of albumin on the kinetics of ascorbate oxidation Evgenia Lozinsky a
a;
*, Artem Novoselsky a , Alexander I. Shames b , Oshra Saphier a , Gertz I. Likhtenshtein a , Dan Meyerstein a;c
Department of Chemistry, Ben-Gurion University of the Negev, 84105 Beer-Sheva, Israel b Department of Physics, Ben-Gurion University of the Negev, Beer-Sheva, Israel c College of Judea and Samaria, Ariel, Israel
Received 27 October 2000; received in revised form 23 January 2001; accepted 25 January 2001
Abstract The fluorescence intensity of the fluorophore in dansyl piperidine-nitroxide is intramolecularly quenched by the nitroxyl fragment. Therefore, the oxidation of ascorbic acid by the fluorophore-nitroxide (FN) probe can be monitored by two independent methods: steadystate fluorescence and electron paramagnetic resonance. Bovine serum albumin (BSA) affects the rate of this reaction. The influence of BSA on the rate is attributed to the adsorption of both ascorbate and the probe to BSA. Adsorption of ascorbate to BSA is confirmed by NMR relaxation experiments. The spatial distribution of the molecules on the BSA surface changes the availability of ascorbate and FN to each other. The results also point out that, in the presence of BSA, the autoxidation of ascorbate is significantly slowed down. The effect is studied at different pH values and explained in terms of the electrostatic interaction between the ascorbate anion and the BSA molecule. ß 2001 Elsevier Science B.V. All rights reserved. Keywords : Fluorophore-nitroxide ; Ascorbate oxidation; Bovine serum albumin
1. Introduction Recently nitroxyl spin labels have been extensively used as redox probes to measure the antioxidant status of biological liquids [1^3]. The only natural antioxidant which has been found to reduce nitroxides is ascorbate, AH3 [4]. Novel improved spin labels with extended properties and possibilities have been recently introduced [5^8]. These compounds contain a £uorophore (F) and a nitroxide (N). An example of this type is the dansyl piperidine-nitroxide used in the present study:
The covalently linked nitroxide radical quenches the £uorescence intensity of the £uorophore; therefore, the electron paramagnetic resonance (EPR) signal decay due
* Corresponding author. Fax: +972-7-647-2903; E-mail : [email protected]
to its reaction with AH3 is accompanied by the simultaneous enhancement of the £uorescence intensity. Thus two independent spectroscopic methods might be used to follow the reaction : steady-state £uorescence and EPR. Naturally, the increase of the £uorescence intensity and the decay of the EPR signal occur with the same rate. The reaction between the FN probe and excess ascorbate obeys a pseudo-¢rst-order rate law. The dependence of the observed rate on ascorbate concentration may be used as a calibration line for the determination of unknown concentrations of AH3 [5]. Thus, the FN probe might be used as a convenient tool for the estimation of the ascorbate content of di¡erent samples. However, attempts to use this calibration line for the measurements in more complicated solutions failed, i.e. the calibration line obtained in bu¡er solutions cannot be applied directly to many samples from living organisms, e.g. plasma and blood. It seemed plausible that this e¡ect is due to the interaction of ascorbate and/or the FN probe with proteins. Since albumin accounts for approx. 60% of the total protein in blood serum with a concentration of about 0.6 mM (40 g/l) [9], its solutions can serve as a model of mammalian plasma. The choice of albumin as a model was also based on the observation that it participates in the transport, distribution and metabolism of many endogenous and exogenous spe-
0304-4165 / 01 / $ ^ see front matter ß 2001 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 1 6 5 ( 0 1 ) 0 0 1 0 0 - 3
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cies. This transport function stems from the very high a¤nity of many organic and inorganic compounds (e.g. fatty acids, amino acids, steroids, metal ions, numerous drugs and dyes) to albumin. Albumin has many, and various binding sites which enable binding of many molecules [10,11]. It was decided therefore to study the in£uence of BSA on the kinetics of the reaction between dansyl piperidinenitroxide and ascorbate (AH3 ). The results indeed point out that both AH3 and FN are adsorbed to BSA and that this adsorption a¡ects the kinetics of the reaction between AH3 and FN. 2. Materials and methods 2.1. Materials Ascorbic acid, amino-TEMPO (4-amino-2,2,6,6-tetramethylpiperidinooxy nitroxide), TEMPOL (4-hydroxy2,2,6,6-tetramethylpiperidinooxy nitroxide) and BSA (fatty acid free) powders were purchased from Sigma. The FN probe was synthesized according to the procedure described in the literature [12], i.e. by mixing the commercial amino-TEMPO with dansyl chloride in pyridine. Stock solutions (0.01 M) of the FN probe were prepared in DMSO :water (3:7) and diluted prior to use. Aqueous solutions of BSA, TEMPOL and ascorbic acid (1 mM and 10 mM) served as stock solutions. The solution of ascorbic acid was kept under N2 before use. 2.1.1. EPR EPR spectra were recorded using a Bruker EMX-220 digital X-band spectrometer equipped with a Bruker ER 4241VT temperature control system. Solutions for analysis were prepared from stock solutions by dilution in PBS. Liquid samples were drawn into 1 mm i.d. glass Pasteur pipettes sealed at the bottom. Spectra at T = 120 K were recorded with 9.40 GHz microwave frequency, 0.20 mW non-saturating microwave power, 100 KHz ¢eld modulation of 0.05 mT amplitude. All spectra at T = 297 K were recorded with the following parameters: 9.40 GHz microwave frequency, 20.12 mW non-saturating microwave power, 100 kHz ¢eld modulation of 0.1 mT amplitude. Kinetics of the decay of the EPR signal of non-immobilized probe molecules were measured by ¢xing the magnetic ¢eld at the top peak position of the high ¢eld hyper¢ne line (M = +1) and recording in the Time Scan mode. Kinetics of the decay of the EPR signal of immobilized probe molecules were measured in the same manner by ¢xing the magnetic ¢eld at the position of the corresponding low ¢eld hyper¢ne line using the Time Scan mode and modulation amplitude 0.5 mT. The total error in the signal amplitude determination during the kinetic measurements was about þ 15%. The time delay between the initial mixing and the start of the scans in every experiment did not
exceed 80 s. Both EPR spectra and kinetics were processed using the Bruker WIN-EPR and Microcalc Origin software. 2.1.2. Steady-state £uorescence Fluorescence emission spectra and kinetics of the steady-state £uorescence increase were recorded using an ISS-GREG90-MM multifrequency phase modulation spectro£uorimeter SLM 4800 with a 450 W xenon lamp. Liquid sample solutions were prepared directly in the cuvette from stock solutions by dilution in PBS. Spectra were obtained using the following parameters : excitation wavelength 360 nm, scale 60%. All other spectral parameters are given in the legends of the ¢gures. The total error in signal amplitude determination during the kinetic measurements was about þ 10%. The data from £uorescence measurements were processed with KaleidaGraph software. 2.1.3. Nuclear magnetic resonance (NMR) 1 H NMR spectra (5 mm sample tubes, 298 K) and the measurement of 1 H T2 relaxation times were performed at 500.1 MHz on a Bruker DMX-500 Fourier transform spectrometer. 2 H2 O was used as an internal lock, and the residual H2 O solvent was used as an internal reference (NH = 4.8). Standard Bruker microprograms were utilized for the CPMG (Carr-Purcell-Meiboom-Gill) technique [13]. The error of the T2 measurement was smaller than þ 10%. 3. Results and discussion In order to check whether the FN probe is adsorbed to BSA, its EPR and steady-state £uorescence spectra in PBS and in BSA containing solutions were measured. Both methods demonstrate that the FN probe is bound to the albumin matrix (Fig. 1A,B). The EPR spectrum shows signi¢cant changes in the line shape attributing to the FN molecules incorporated into BSA (see additional signals marked by arrows in Fig. 1A). The shift of the £uorescence maximum to the blue points out that the binding occurs via the £uorophore fragment. Other EPR experiments were done to elucidate whether the FN probe is also bound to BSA through its nitroxide fragment. Parallel hyper¢ne splitting 2A0zz obtained from the spectrum of frozen solution is known to be a measure of the local polarity. We measured the 2A0zz values from the EPR spectra of FN in frozen bu¡er solution (at T = 120 K) in the presence and absence of BSA and found them to be the same: 2A0zz = 7.4 mT. It may be concluded that the nitroxide fragment has no a¤nity to the protein matrix and therefore most probably at room temperature it freely rotates in the aqueous phase. Such a conclusion is corroborated by the observation of EPR spectra of TEMPOL, which is an analogue of the radical fragment of FN. Nei-
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Fig. 1. Binding of FN (0.1 mM) to BSA. (A) EPR spectra of FN in PBS (999) and BSA (0.5 mM) (9 9 9) solution. Arrows show the signals of the immobilized probe. The spectrum in BSA solution was recorded at a 4-fold receiver gain. (B) Fluorescence emission spectra of free FN (0.1 mM) in PBS (999) and in solutions containing various concentrations of BSA (9, 0.01 mM; - - -, 0.05 mM; - 999 -, 0.1 mM). Emission and excitation slits 16 nm, scan rate 10 nm/s. All measurements were conducted at pH 7.4.
ther room nor low temperature spectra are a¡ected by the addition of BSA to its solutions, i.e. TEMPOL is not adsorbed to BSA. Like most small negatively charged organic molecules, ascorbate has a very high a¤nity to BSA, as demonstrated by a dialysis experiment [14]. In order to obtain a better understanding of the type of interaction between ascorbate and BSA, the NMR transverse relaxation times T2 of the
ascorbate protons in 2 H2 O and in solutions containing BSA were measured. The results for the methyl-ol proton (marked by `b' in Fig. 2) are summarized in Table 1. The decay of the transverse magnetization in pure 2 H2 O ¢ts a single exponent, with T2 = 765 ms. However, in the presence of BSA, the decay can be ¢tted only by the sum of two exponents. Binding of the ascorbate molecule to the massive protein causes a drastic decrease in the T2 of the
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Fig. 2. NMR spectrum of vitamin C (5 mM) after overnight incubation. Solvent 2 H2 O, pD 3.8.
ascorbate protons. The values of T2 for the two components (bound and free) are practically independent of the BSA content, while their ratio varies. The results point out that vitamin C is not bound tightly to BSA since the NMR lines of protons of the bound molecules are still observed. Moreover, all the ascorbate protons observed in the NMR spectrum relax with di¡erent rates, indicating that the mobility of the methine and methylene groups of the bound molecules di¡ers (Table 2). In order to estimate the contribution of BSA to the solution viscosity, the same experiment was performed with acetone, a small organic molecule that does not bind to albumin and possesses a very long T2 . The decrease in T2 of the acetone protons was found to be less than 20% from the initial value at 0.25 mM BSA concentration, while for ascorbate protons the decrease in T2 is 8.5^60-fold for the bound molecules at the same BSA content. These results show that the shortening of the transverse relaxation time is caused mainly by the binding of the ascorbate to albumin. Moreover, in the case of acetone the decay of the transverse magnetization always ¢ts a single exponential and is practically independent of the albumin content. The experimental results (Table 1) demonstrate that about 100 molecules of ascorbate can be adsorbed to one molecule BSA. This conclusion di¡ers from that reported in the literature Table 1 Percentage of bound and free components of ascorbate (5 mM) in BSA containing solutions and in the absence of BSA [BSA] (mM) Component 1 (765 ms) (%) Component 2 (90 ms) (%) pD 3.8.
0
0.001
0.01
0.05
0.025
100 0
87 13
85 15
68 32
10 90
where it was suggested that ten is the maximal number of the bound ascorbate molecules for one molecule of BSA [14]. In the presence of BSA, the transverse relaxation times of the ascorbate protons are pH-dependent (Fig. 3). The results clearly point out that T2 is shortest at pH 5.0 and becomes longer at both pH 3.8 and 7.0. As the ¢rst pKa of ascorbate is AH2 H2 O1AH3 H3 O pK a 3:8
1
and as AH3 is expected to be bound stronger to BSA, i.e. K1
AH3 BSAAH3 ÿ BSA
2
K2
AH2 BSAAH2 ÿ BSA
3
K1 s K2 , one expects an increase in the binding when the pH is raised from 3.8 to 5.0. The observed decrease at pH 7.0 is attributed to the isoelectric point of albumin at pH 5.2 [15]. The binding of AH3 to the negatively charged BSA at pH 7.0, although naturally positively charged substituents still are present on BSA at this pH, is clearly weaker. The reaction between the FN probe and AH3 was investigated by two independent techniques: EPR and Table 2 Values of T2 for free (component 1) and bound (component 2) ascorbate protons Proton (ppm) Component 1 (ms) Component 2 (ms)
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a(4.84)
b(3.97)
c(3.66)
820 60
765 90
245 4
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Fig. 3. Decay of transverse magnetization of the ascorbate proton (marked as `b' in Fig. 2) at di¡erent pD values (E, 3.8; tion of ascorbic acid and BSA 5 mM and 0.05 mM, respectively. Solvent 2 H2 O.
steady-state £uorescence. In the presence of excess ascorbate, the kinetic curves obey a ¢rst-order rate law. The observed rates, kapp , are proportional to [AH2 ] (Fig. 4). Addition of up to 1 mM BSA to the solution accelerates the observed rate. The e¡ect of BSA on the rate of the reaction has a clear concentration-dependent character (Fig. 4). From the slopes of the lines in Fig. 4 the rate constants for the reaction of ascorbate with FN (remembering that two FN molecules are reduced by each ascor-
57
a,
5.0; O, 7.0). Concentra-
bate and assuming that the ¢rst reaction is the rate determining step) are calculated. In the presence of 0.1 mM and 0.8 mM BSA, the rate constants of the reaction were found to be 25, and 38 M31 s31 respectively, while k = 13 M31 s31 in the absence of BSA. At low concentrations of BSA, the e¡ect of albumin on the rate constant is insigni¢cant. At higher concentrations the reaction is accelerated; however, not all FN molecules are reduced though [AH3 ] s [FN]. At 1 mM and higher concentra-
Fig. 4. Dependence of the observed rate of FN (0.1 mM) reduction on ascorbate concentration at di¡erent concentrations of BSA (a, no BSA; mM ; O, 0.8 mM). EPR measurements. PBS (pH 7.4); T = 297 K.
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E,
0.1
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Fig. 5. E¡ect of pH on the rate of the reaction of FN (0.1 mM) with ascorbate in the presence of 0.1 mM BSA. O, pH 4.0;
tions of BSA, the EPR signal decay or/and the £uorescence enhancement processes are too fast to be measured in the CW experiment. A large portion of the FN which, according to its EPR spectrum, is all bound to BSA, is long lived in the time scale of the experiments: t1=2 s 10 min. Dilution of this solution results in the reappearance of the measurable kinetics: the EPR signal starts to decay and the £uorescence intensity grows. It should be noted that the reaction of TEMPOL with ascorbate, as followed by the EPR technique, is not a¡ected by the presence of BSA. The high resolution of the EPR spectrum of FN in BSA containing solutions enables the recording of the decay of the immobilized component as well as the free one. In all cases, the immobilized and the free components decay with the same rate constant, though as stated above a part of the immobilized FN remains at the end of the reaction. This result points out that in the system three components of the FN probe are present: (1) not adsorbed, free FN in the solution ; (2) loosely bound FN to BSA; this fraction is in fast equilibrium with the free FN, and therefore their rate of reaction with ascorbate is equal ; (3) tightly bound FN, which reacts slowly with the ascorbate either inherently or due to a low exchange rate with the other types of the probe. The observations can be explained by the spatial distribution of ascorbate and FN, which are adsorbed to BSA. When BSA is present at low concentrations, molecules of FN and ascorbate bind to the same BSA molecule. The sites of the primary binding are close; therefore, the probability of collision increases and the reaction proceeds faster. At high concentrations of BSA, the number of
a,
pH 5.0;
E,
pH 7.0.
available sites on BSA for FN and AH3 is large. A part of the FN and AH3 molecules are located at remote sites of BSA or are even bound to di¡erent BSA molecules. Thus they are separated from each other in space and the reaction is slowed down. This explanation is supported by the observation that the percentage of FN consumed in the reaction decreases with the increase in the concentration of BSA. The EPR spectrum of the residual amount of FN is observed after the reaction is over, though excess of ascorbate is present in the solutions. At 1 mM BSA, almost 100% of the FN remains bound to albumin and the reaction of these FN molecules with ascorbate is not observable in the time scale of the measurement (20 min). It was observed that even at relatively low concentrations of BSA (0.01^0.1 mM), a weak EPR signal of bound residual FN exists. At 0.1 mM BSA it achieves about 30% of the FN added. The estimation of the exact content of the rest of the FN at lower BSA concentrations is di¤cult because of the weak EPR signal and, correspondingly, low signal/ noise ratio. As the e¡ect of BSA on the rate of the reaction between FN and ascorbate is attributed to their binding to BSA, this e¡ect is expected to be pH-dependent. Indeed, such a dependence is observed ; the observed rate constants are 14, 87 and 42 M31 s31 at pH 4.0, 5.0, and 7.0, respectively (Fig. 5). The results are consistent with the pH dependence of the ascorbate binding to BSA (Fig. 3). The e¡ect of pH on the rate of reaction of FN with ascorbate in the absence of BSA was measured in bu¡ered solutions; the observed rate constants are 11, 19 and 14 M31 s31 at pH 4.0, 5.0 and 7.0 respectively. The small decrease in the rate between pH 5.0 and 7.0 is attributed to the pKa of the amino group of the probe.
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Fig. 6. Binding of FN (0.1 mM) to BSA. The binding ratio was calculated using the formula Ratio = (133DIN3 /DIN)U100%, where DIN is the normalized double integral of the whole EPR signal and DIN3 is the normalized double integral of the high-¢eld non-immobilized component, which has only a negligible overlap with the signal of the bound FN. O, pH 4.0; a, pH 5.0; E, pH 7.0; U, pH 7.4.
These results clearly demonstrate that the major factor a¡ecting the rate of the reaction between FN and vitamin C in the presence of BSA is the binding of both AH3 and FN to BSA. The e¡ect of pH on the binding of FN to BSA was also measured as another plausible contribution to the kinetics of ascorbate oxidation by FN. The results presented in Fig. 6 indicate that this binding increases from pH 4.0 to 5.0, probably due to the pKa of the amino
group of the FN. Above pH 5.0 only minor e¡ects are observed. The reaction of ascorbate with FN can also be used to follow the e¡ect of BSA on autoxidation of ascorbate. Ascorbate is oxidized by dioxygen in homogeneous neutral aqueous solutions [15]. In the presence of several proteins, including BSA, the rate of this process is signi¢cantly slowed down. Indeed, the EPR signal of FN does not
Fig. 7. Protective e¡ect of BSA on the kinetics of ascorbate autoxidation. Observed rate of FN (0.1 mM) reduction in a freshly prepared solution (a) and in a solution incubated overnight containing ascorbate and BSA (F) vs. BSA concentration. Concentration of ascorbate 0.1 mM. pH 7.4; T = 297 K.
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disappear when the probe is added to an ascorbate solution which was kept overnight at free access to dioxygen. Further addition of FN to an aerated solution of ascorbate and BSA which was kept for 14 h results in an EPR signal decay, pointing out that the ascorbate was preserved against autoxidation (Fig. 7). The NMR spectra of ascorbate, kept overnight in the presence and absence of albumin, also point out that BSA protects AH3 from autoxidation. In the absence of BSA the NMR of dehydroascorbate is clearly observed (Fig. 2). When the solution of AH3 contains BSA the peaks of the dehydroascorbate do not appear. 4. Concluding remarks The use of the FN probe, and probably other probes, for the determination of the ascorbate concentration in biological systems which contain proteins is hindered due to the protein concentration-dependent interactions between the proteins and/or the ascorbate and the probe. The addition of BSA to a mixture of FN and ascorbate a¡ects the kinetics of the reaction in a concentration-dependent fashion. Two, in principle opposing, e¡ects are observed: (1) the rate of the reaction is accelerated with increasing BSA concentration; (2) however, with the increase in BSA concentration a large part of the probe becomes `unreactive', though excess ascorbate is present in the solution. These e¡ects are attributed to the adsorption of both the probe and the ascorbate to the protein thus (a) bringing them closer to each other at low protein concentrations and (b) separating them spatially at high BSA concentrations. The binding of ascorbate to BSA is shown to be pHdependent due to two reasons: (1) the pKa of ascorbate; (2) the total charge of the BSA molecule. The electrostatic interaction between ascorbate and the protein decreases considerably the rate of its autoxidation.
References [1] N. Kocherginsky, H.M. Swartz, Nitroxide Spin Labels, CRC Press, Boca Raton, FL, 1995, pp. 114^172. [2] G. Likhtenshtein Spin Labeling Methods in Molecular Biology, Wiley-Interscience Press, 1982. [3] P.A. Motchnik, B. Frei, B.N. Ames, Measurement of antioxidants in human blood plasma, Methods Enzymol. 234 (1994) 269^279. [4] B. Frei, L. England, B.N. Ames, Ascorbate is an outstanding antioxidant in human blood plasma, Proc. Natl. Acad. Sci. USA 86 (1989) 6377^6381. [5] E.M. Lozinsky, V. Martin, T. Berezina, A. Shames, A. Weis, G.I. Likhtenshtein, J. Biophys. Biochem. Methods 38 (1999) 29^42. [6] S. Pou, Y.I. Huang, A. Bhan, V.S. Bhadti, R.S. Hosmane, S.Y. Wu, G.L. Cao, G.M. Rosen, A £uorophore-containing nitroxide as a probe to detect superoxide and hydroxyl radical generated by stimulated neutrophils, Anal. Biochem. 212 (1993) 85^90. [7] S.E. Herbelin, N.V. Blough, Intramolecular quenching of excited singlet states in a series of £uorescamine-derivatized nitroxides, J. Phys. Chem. B 102 (1998) 8170^8176. [8] S.A. Green, D.J. Simpson, G. Zhou, P.S. Ho, N.V. Blough, Intramolecular quenching of excited singlet by stable nitroxyl radicals, J. Am. Chem. Soc. 112 (1990) 7337^7346. [9] B. Halliwell, Albumin ^ an important extracellular antioxidant?, Med. Pharmacol. 37 (1988) 569^571. [10] X.M. He, D.C. Carter, Atomic structure and chemistry of human serum albumin, Nature 358 (1992) 209^214. [11] D.C. Carter, X.-M. He, S.H. Munson, P.D. Twigg, K.M. Gernert, M.B. Broom, T.Y. Miller, Three-dimensional structure of human serum albumin, Science 4 (1989) 1195^1198. [12] G.I. Likhtenstein, V.R. Bogatyrenko, A.V. Kulikov, K. Hideg, H.O. Hankovsky, N.V. Lukoianov, A.I. Kotelnikov, B.S. Tanaschelchuk, Study of superslow motion in solid solutions by the method of physical probes, Dokl. Akad. Nauk SSSR 253 (1980) 481^484. [13] T.C. Farrar, E.D. Becker, Pulse and Fourier Transform NMR. Introduction to Theory and Methods, Academic Press, New York, 1971. [14] V.C. Okore, E¡ect of ascorbic acid on the binding of cloxacillin sodium to bovine serum albumin, Arzneim.-Forsch. Drug Res. 44 (1994) 671^673. [15] Y. Wen, P. Dubin, Potentiometric studies of the interaction of bovine serum albumin and poly(dimethyldiallylammonium chloride), Macromolecules 30 (1997) 7856^7861.
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