Elecwochimica Acta, Vol. 39. No. 16. pp. 2455-2460. 1994 Copyright 0 1994 Ekvier Bcience Ltd. Ptintcd in Great Britain. All tights moved @x3-4686/?% $7.00 + 0.00
STUDY ON THE KINETIC PROCESS OF THE COMPLEX REACTION BETWEEN BILIRUBIN AND CYCLODEXTRINS JIANJUNNIU, GUANGJIN CHENGand SHAOJUNDONG* Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Academia Sinica, Changchun 130022, People’s Republic of China (Received 23 September 1993) Abstract-The complex behavior of bilirubin (BR) with /?-CD (cyclodextrin) and y-CD in aqueous and dimethylformamide (DMF) solution was investigated by absorption spectroscopy and cyclic voltammetry, respectively. The data shows that the complexation mechanisms in these two solvents are different. In aqueous solution, the complexes of BR$-CD and BR-y-CD were formed by internal inclusion reactions, while in DMF, they were formed by external, non-inclusion ways. The dissociation constants obtained for these complexes are K,, = 9 5 x 10T4 M, K,, = 5.1 x 10e4 M in aqueous solution and K,, = 12.5 x 10m2M, K,, = 1.73 x IO-‘M in DMF, respectively. Key words: bilirubin, cyclodextrin, complexation, bioelectrochemistry, spectroelectrochemistry.
INTRODUCTION As a result of the truncated
cone shape, with many hydroxyls locating on the narrow and wide rims of the molecule, the external face of cyclodextrin (CD) is hydrophilic, while the internal cavity, the wall lined with hydrogen and glycosidic oxygen bridge atoms, is hydrophobic[l]. This property of CD makes it possible for the hydrophobic guest molecule to enter its cavity and be held tightly in this special micro-environment to undergo particular reactions. This shows that CD not only serves as an excellent model of the artificial enzyme, but also has simple and stable characteristicsC2, 33. For its unusual structure and the derived special functions. CD has amply been used in medical and food industries, organic synthesis, chemical luminescence, environment protection, etc[4-93. In recent years, the most attractive application of CD has been in chromatography as chiral mobile phase additives or as a chiral stationary phase for direct enantiomeric or isomeric separations[lO, 111, in which the selective inclusion and chiral recognition functions of CD are utilized. Stereoselective complexation between CD and an enantiomer of some biomolecules leads to changes in the optical characteristics of these molecular solutions, which have received more and more attention in the study of biochemical reactions. LeBas et al. first noted that the external compensated aqueous solution of BR exhibited clear circular dichroism in the presence of CD, indicating the occurrence of complexation between these two molecules. Similar to the reactions of BR with other chiral reagents, such as albumin and some organic bases, this complexation involves stereoselective process, from which the conformational * To whom correspondence should be addressed.
equilibrium between two enantiomers of BR is perturbed and the circular dichroism is induced. The chiral selective complexation is also important in biological reactions of BR enzymic glucuronidation. Though the complex reaction between BR and CD is found in aqueous solution by circular dichroism, the nature and the kinetic process of the complexation have not been investigated yet. In the present work, the complex reactions of BR with /?-CD and y-CD, in aqueous solution and in the organic solvent dimethylformamide (DMF), are studied by absorption spectroscopy and cyclic voltammetry, respectively. Different mechanisms of the complex reactions in these two solvents are proposed and the corresponding dissociation constants are determined.
Bilirubin with a purity of 99% was obtained from Sigma. 0.1 M Tris buffer (pH 9.0) was prepared from Tris base and hydrochloric acid. DMF, analytical grade, was distilled from calcium hydride before use and kept in a desiccator. Tetrabutylammonium perchlorate (TBAP) purchased from Fluka was used as the supporting electrolyte. The daily prepared stock solution of BR was protected from light and kept in a refrigerator. Other reagents were all analytical grade. Instrumentation
All the measurements of absorption spectra were carried out on a DMS-90 spectrophotometer. The electrochemical experiments were performed with a
PAR model 173 potentiostat and a model 175 universal programmer. The electrochemical cell was a three-electrode system, a platinum wire (diameter 3.0mm) sealed in a soft glass tube used as a working electrode (WE), a platinum foil as an auxiliary electrode (AE), and Ag/AgCl (1.0 M KCl) as a reference electrode (RE). Before each electrochemical measurement, the testing solution was accurately deoxygenated by N, (99.999%). The WE was first polished successively with 0.5, 0.3 and O.OSpm o-Al,O,, then washed twice by ultrasonication in twice-distilled water, dried thoroughly and used in the experiments. All the experiments were performed at room temperature and under a nitrogen atmosphere.
RESULTS AND DISCUSSION Complex behaviors of BR with B-CD and y-CD in aqueous solution and determination of the dissociation constants
It is known that the formation of inclusion complexes with CD leads to changes in the absorption spectra of a variety of organic molecules. From this phenomenon, both the complex behavior and the kinetic constant of the host-guest inter-reactions can be evaluated. Curve (a) in Fig. 1 gives the absorption spectrum of BR in a pH 9.0 Tris buffer solution with a single maximum absorbance at 432 nm. By holding
the concentration of BR at 2.0 x lo-’ M and adding /l-CD into the solution, the spectrum varies remarkably. Its absorbance intensity decreases and absorption peak becomes broad correspondingly (see Fig. 1). The appearance of two isosbestic points at about 335 and 487 nm indicates that the 1: 1 complex between BR and /?-CD is formed. This conclusion is consistent with the result of circular dichroism[l2]. The inclusion complex process of BR with y-CD is similar to that with &CD. Figure 2 shows the effect of y-CD at various concentrations (same as in Fig. 1) on the BR absorption spectrum. It can be seen that the decreasing degree of absorbance intensity with the addition of y-CD is larger than that of /I-CD, which demonstrates that the inclusion complex of BR-y-CD is more stable than BR-b-CD. From the absorption spectra changes for BR by the addition of B-CD or y-CD, the dissociation constants of the complex reactions can be determined. Suppose [CD,] and [BRc] are the total concentration of CD and BR, respectively, AAbs is the change in absorbance by the addition of /I-CD or y-CD, K, is the dissociation constant, and AE is the difference in the molar absorptivities for free and complexed BR, then we have[ 171
Fig. 1. Spectrum of 2.0 x low5 M BR at varying /&CD concentrations. Solvent is aqueous solution, Tris buffer (pH 9.0). /?-CD con-tration (M): _(a) 0.0, (b) 5.0 x !O-*, (c) 1.0 x lo-“, (d) 3.0 x IO-” and(e) 5.0 x lo-“.
CCW + CBRJ ’
Under the conditions of [CD,][BR,] s Cz (C is the concentration of the complex of BR-/I-CD or BR-yCD), the plot of [CD,][BR,]/AAbs vs. ([CD,,] + [BR,]) is a straight line, with a slope of l/A& and an intercept
Fig. 2. Spectrum of 2.0 x lo-’ M BR at varying y-CD concentrations. Solvent is aqueous solution, Tris buffer (pH 9-O), y-CD conc+ntration (M): !a) 0.0, (b) 2.0 x !O-•, (c) 5.0 x 10-‘,(d) 1.0 x lo-’ and(e) 5.0 x lo-‘.
Complex reaction between bilirubin and cyclodextrins In Figs 1 and 2 are plotted the diagrams of [CD,][BR,]/AAbs vs. ([CD,,] + [BR,]) for two complexes of BR-/?-CD and BR-y-CD at 432nm. Two straight lines with linear coefficients rna+cu = 0.9996 and rBR_,,_cD = 0.9988 are obtained, respectively (see Fig. 3). From the intercepts, the dissociation constants of BR-B-CD and BR-y-CD, K,, = 9.50 x low4 M and K,, = 5.05 x 10e4 M are determined. Both the values of K,, and K,, are small, showing that the two inclusion complexes formed are stable. However, K,, is almost twice as large as K which further confirms that y-CD has a larger mdo2ldcularcavity with more inclusion ability with BR than /?-CD. Complex behavior in DMF
The investigation of the complex reaction of BR and CD in DMF is convenient with electrochemical techniques, because BR is an electroactive species, and both BR and CD have a relatively high solubility in this solvent. Figure 4 gives the cyclic voltammograms (cv) of BR in DMF solution. It shows a couple of quasi-reversible redox peaks (the dotted line). In the absence of B-CD, the cathodic and
anodic peak potentials, E and Ep , are at - 0.72 and -0.60 V (vs. AgJAgClr respecttvely, resulting in the peak-to-peak separation AE, = 0.12V. When B-CD is added to the solution, E,, and E, shift to negative and positive separately, and the AE, increases to 0.22 V, which indicates that the irreversibility of the redox process increases. Meanwhile, the peak currents also increase in the presence of B-CD (the real line in Fig. 4). The effect of y-CD on the cv curve of BR is given in Fig. 5. This figure also shows the shift in the peak potentials and the increase in peak currents. These potential and current changes caused by the addition of /?-CD or y-CD demonstrate that the complexes of BR-/I-CD and BR-y-CD are also formed in organic solvent DMF, so the kinetic processes of these complexations can be investigated by the electrochemical method. For a complex of the electro-active substance 0 with CD, the electrochemical reactions can be divided into two steps: O--CD+0
0 + ne-+R.
Suppose i, and i,, are the reduction current of 0 in the presence and absence of CD, respectively, then we have[ 191 i2 = * (ii, - iz) + ii,, - CD, ’ CCDI
where K, is the dissociation constant of the complex and [CD] is the concentration of CD in solution. According to equation (4), i,, and the different values of i, are determined respectively by holding the concentration of BR constant and varying the concentration of /I-CD or y-CD. The diagrams of is =
([BR) + [CD)) x10-‘/M
Fig. 3. The plots according to equation (1) for the interactions between BR and (A) /?-CD, and (a) y-CD.
Fig. 4. The cyclic voltammogram of 2.0 x 10e4M BR in DMF: (. . .) in the absence of P-CD; (-) in the presence of 1.2 x lo-‘M B-CD.
EIV vs. AglAgCl Fig. 5. The cyclic voltammogram of 2.0 x 10e4M BR in DMF: (. . .) in the absence of y-CD; (-_) in the presence of 1.2 x lo-* M y-CD.
JIANJUNNW et al.
.* \ '\ \
\_ 1 53
Fig. 6. The plot for current analysis for the complexation between BR and b-CD. Solvent is DMF.
of BR-B-CD and BR-pCD are plotted and two straight lines, shown in Figs 6 and 7, are obtained. From the slopes, the dissociation constant of BR+CD, K,, = 12.5 x 10e2M, is obtained. This large value of K,J shows that the complex of BR+CD formed in DMF is not very stable. The K,, obtained for BR-p CD is 1.73 x lo-‘M, which is much smaller than K,, . This result indicates that, in DMF, the complex ability of BR with y-CD still vastly exceeds that with /?-CD.
vs. (ii,, - i,2) for the complexes
Fig. 7. The plot for current analysis for the complexation between BR and y-CD. Solvent is DMF. The difirent complex processes in two solvents
From the results above, we have determined that the complexes with different stabilities between BR and /?-CD or y-CD can be formed in both aqueous and DMF solvents, but is is noticed that the phenomena of complexations in these two solvents are
(4 Fig. 8. The proposed complexing mechanism of BR with /I-CD and with y-CD in basic aqueous solution.
Complex reaction between bilirubin and cyclodextrins
quite different. In aqueous solution, as shown in Figs 1 and 2, the absorption spectrum of BR is greatly changed by the addition of B-CD or y-CD, while in the DMF solvent no spectrum change can be seen when we operate the same experiments as in the aqueous solution. This difference in optical properties indicates that the complex behavior between BR and CD in DMF and in aqueous solution are not the same. Two types of configurational complexes may be formed by different combining mechanisms. The circular dichroism of BR in buffered aqueous solution shows that /I-CD prefers to select the left enantiomer to form the inclusion complex, which causes changes of optical activity of the BR solution and exhibits the induced circular dichroism Cotton effects (CEs). However, the appearance of the CEs closely relates to the pH values. The strong CEs can only be seen in the basic solutions, while in neutral or acid solutions, its CEs are very weak or disappear altogether. The variation tendency of the absorption spectra of BR is similar to that of circular dichroism in the presence of /?-CD or y-CD. The big changes in the spectra only occur at about pH > 8. These variations of circular dichroism and absorption spectra demonstrate that the chiral selective inclusion complexation between BR and /LCD or y-CD can only be achieved in basic conditions. In neutral aqueous solution BR keeps a rigid structure (see Fig. 8a) by six tightly linked intramolecular H bonds. The high steric inhibition of this conformation makes the inclusion complexation between BR and CD impossible. When the basicity of the solution is increased, acid dissociation of BR occurs, and the intramolecular H bonds begin to break up. Total dissociation (two carboxyls dissociate first, and two lactams at higher pH) is achieved at pH > 8; the intramolecular H bonds no longer exist under this condition, resulting in the conformational transformation of BR from the rigid structure to a loose chain one. It is known from the above results that the greatest variations of circular dichroism CEs and the absorption spectra induced by /?-CD or y-CD occur in the same condition, showing that the complexed structure of BR with CD is a dissociated one in basic aqueous solution (see Fig. SC).The proposed processes of the complexation are shown in Fig. 8. For the transformation of the conformation of BR by acid dissociation, and the flexible chain structure generated, the steric inhibition for inclusion complexation with CD is greatly decreased. Moreover, the rotation about the C-C single bonds between C&,, and C,,-C,, make BR C,-C,, c,,-c15, adjust to the best angle to enter the molecular cavity of P-CD or y-CD, from which the inclusion complexes are formed. The rotation of the above C-C bonds can also make the two carboxyls of BR to the hydroxyls at the rims of CD as close as possible to form the extra H bonds, which increases the stability of the complexes. The cavity of y-CD is larger than y-CD, so BR can enter the cavity more easily and deeply, the distance between the carbonyls of BR and the hydroxyls of CD then becomes smaller, resulting in stronger extra H bonds and smaller K, value of the complex BR-yCD than that of BR-/3-CD. In these stereo-inclusion
complexes, a part of the chromophore of BR enters into the internal cavity of CD, leading to a change in its electron environment and resulting in the obvious variations of the absorption spectra. In the DMF solvent, it is difficult for BR to undergo the acid dissociation, so it keeps the rigid structure, which demonstrates that the complex conformation of BR in DMF is not the dissociated form, as in the aqueous solution, but the rigid one. Comparing the K, values of BR-/?-CD and BR-y-CD in DMF and in aqueous solution, we know that the dissociation constants in DMF exceed those in aqueous solution by one or two orders of magnitude. The great difference in these two solvents shows that the complexation of BR and CD is no longer in the very stable host-guest form by internal inclusion, as in aqueous solution, but in the non-inclusion form by external complexation, which may be performed by the extra H bonds between the rim hydroxyls of CD and the carboxyls of BR. The proposed structure of the complex is shown in Fig. 9. Because BR has six intramolecular H bonds itself, the combining force of these complex H bonds is relatively weak, which leads to the great decrease of the stabilities and the large increase in the K, values. For the reason that BR cannot enter into the cavity of CD in DMF and the relatively weak complex force between these two molecules, as shown in Fig. 9, the effect of the micro-environment of the pyromethenone chromophores of BR by the formation of the complexes is very small. Therefore, the absorption spectrum of BR in DMF is not changed by the addition of B-CD or y-CD. In the
Fig. 9. The proposed structure of the complexes of BR-/?CD and BR-y-CD in DMF.
JIANJUNNIU et al.
cu investigations, however, the redox reactions of BR are hindered to some extent by the adsorption of CD on the electrode surface. This causes the negative and positive shift of the cathodic and anodic peak potentials, respectively. The reversibility of the redox process then decreases. Also for this effect of adsorption, the concentration of BR at the electrode surface is increased by the formation of the complex between BR and CD, resulting in the increase of the cathodic and anodic peak current in the CDcurve (Figs 4 and 3. Acknowledgement-This project is supported National Natural Science Foundation of China.
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