Shen Xingcan, Liang Hong, Jiang Zhiliang, He Xiwen^#, Shen Panwen ^#
(Institute of Bioinorganic Chemistry, Department of Chemistry, Guangxi Normal University, Guilin 541004,China ^#Department of Chemistry, Nankai University, Tianjin 300071,China)

Received Feb. 10, 2000; Supported by the National Natural Science Foundation of China (NO. 29961001) and the Foundation for Talents Striding across the Century of Guangxi.

Abstract The binding equilibrium between I^- and human serum albumin (HSA) or bovine serum albumin (BSA) has been studied by resonance light-scattering (RLS), fluorescence spectrometry and equilibrium dialysis. The enhancement of RLS were founded as the I^- binding to the HSA or BSA, while the fluorescence quenching at 350 and 700 nm. From the equilibrium dialysis results, the binding of I^- to HSA or BSA fits to a phase distribution model instead of the Scatchard model, and the order of magnitude of phase distribution was found to be 10⁴. The dialysis at different pH indicates that the binding mechanism is probably due to the electrostatic forces between the I^- and protonated amino-acid residues.
Keywords serum albumin, iodine, equilibrium dialysis, resonance light-scattering, phase distribution

1. INTRODUCTION 
Iodine is one of living element, and is also called intellect element ^[1]. The serum albumin, which constitutes 60% of blood plasma, has very important physiological functions. It is, therefore, important to know the detail of I^- interactions with serum albumin, The metal ions, little molecules and medicines etc interacting with HSA or BSA have been reported ^[2-5], but there is no reports about behavior of I^- binding to HSA or BSA. The RLS ^[6], which is a new method put forward recently, has rarely been studied in bioinorganic chemistry and photochemical study of HSA and BSA ^[7]. The investigations of I^- binding to HSA or BSA with RLS, fluorescence spectrometry and equilibrium dialysis are helpful for us to understand absorbing, conveyance, etc physiological function of iodine. Our study indicates that RLS is a sensitive way in bioinorganic chemical study besides ultraviolet and fluorescence spectra.

2. EXPERIMENTAL
Electrophoretic pure HSA and BSA were purchased from Sino-American Biotechnology Company Beijing Branch and mailed freshly. All solutions were prepared with analytical grade reagents and double deionized water. The RLS data were obtained by scanning the excitation and emission monochromators (Dl =0.0 nm) with a shimadzu RF-540 spectrofluorophotometer (Kyoto Japan), within the wavelength region from 200 nm to 1000 nm. The excitation and emission slits were set to 10 nm. With l_ex=296 nm, scanning the emission wavelength region from 200 nm to 1000 nm, the fluorescence spectra of HSA or BSA binding with I^- were obtained. Hitachi UV-3400 spectrophotometer was employed for the concentration of HSA and BSA ^[2]. The steps of equilibrium dialysis were described in ref. [2,5], but were performed in the darkness. The concentration of KI solution before dialysis was titrated with iodimetry, and the concentration of free I^- ions ([I^-]) after dialysis was obtained by spectrophotometry ^[8].

3. RESULTS AND DISCUSSION
3.1 RLS and fluorescence spectra
Fig.1 shows the RLS spectra of I^- binding to HSA. The spectral characteristics of BSA are similar to that of HSA ^[7]. There are five characteristic peaks in the RLS of HSA or BSA, two strong intensity peaks at about 296 nm and 470 nm, a broad peak at about 690 nm, two shoulder peaks at 590 nm and 900 nm respectively. The intensities of the RLS (I_RLS ) are enhanced greatly after I^- binding to HSA. The I_RLS is directly proportional to the ratio of [I^-]/[HSA] in the range of 0-120 at the most sensitive wavelength 296 nm. The concentration of HSA is 1×10^-5 mol / L.

	Fig.1 The resonance light-scattering (RLS) of HSA 1:HSA; 2:[I-]/[HSA]=5:1; 3:[I-]/[HSA]=50:1; 4:[I-]/[HSA]=100:1.  The concentration of HSA is 1×10-5 mol / L.
	Fig.2 Fluorescence spectra 1:HSA; 2:[I-]/[HSA]=5:1; 3:[I-]/[HSA]=50:1. The concentration of HSA is 1×10-5 mol / L.

    The fluorescence spectra are shown in Fig.2. The intensity of fluorescence spectra (I_F ) was quenched greatly at 350 nm and 700 nm which are the first and second fluorescence emission peaks respectively.^[7] The quenching results from the "heave atom effect" of iodine^[9]. Whereas the behavior of the peaks at 296 nm, 592 nm and 888 nm are different from fluorescence emission peaks at 350 nm and 700 nm: as I^- binding to HSA they increase instead quench. The three peaks are the first, second, and third resonance scatter peak ^[7], and their behavior and mechanism is similar to that of RLS ^[10]. These peaks ignored in fluorescence spectra are of significance to find out the information of resonance scattering.

	Fig.3 vs [I-] The concentrations of BSA a:1×10-4; c: 2×10-4 mol/ L; The concentrations of HSA b:1×10-4; d: 2×10-4mol/ L. At pH7.43
	Fig.4 vs pH     The concentrations of BSA and HSA 1×10-5 mol/ L

3.2 Equilibrium dialysis study
The results of equilibrium dialysis at pH7.43 are shown in Fig.3. By plotting vs [I^-], straight lines are obtained, which is not similar to the Cu²⁺, Mn²⁺ etc binding to HSA and BSA ^[2,5]. The ratio /[I^-] ( is the average binding number ^[2]) is practically constants, so the binding of I^- here does not seem appropriate for the Scatchard model which has been widely employed to the combination of small ions or molecules with proteins ^[4,5,11]. Instead a phase distribution model allows a good quantitative treatment of the experimental findings. So the HSA and BSA considered as microphase distinct from the aqueous solution in which it is dispersed. Thus I^- is distributed between the albumin microphase and aqueous solution. The phenomenon is singular ^[3]. The distributed constant coefficient k_dc of I^- binding to HSA and BSA are 2.01×10⁴ L/ mol and 2.31×10⁴ L / mol respectively.
3.3 The pH effect and binding mechanism
Series of equilibrium dialysis experiments of I^- binding to HSA and BSA at different pH are investigated, and the result is shown in Fig. 4. The curves of _(I-₎ vs pH are found there very similar to the curve ^[12] of _(H+) vs pH. The isoelectric point of HSA or BSA is at pH4.7 ^[13]. As pH<4, the lower pH is, the more H⁺ ions bind to serum albumin, and the more positive charge the albumin molecules own ^[12]. Form the Fig.4, the number of binding I ^- increases fast as pH<4. On other hand, when pH>6, the higher pH is, the fewer H⁺ ions bind to serum albumin, and the more negative charge the albumin molecules show, and the fewer I ^- ions bind. In the range of pH 4-6, the two curves change mildly. The results imply that the number of H⁺ binding to amino-acid residues at albumin molecules has an effect to the binding of I^-. As a result that the distributed constant coefficient k_dc is change with pH. It is concluded that I ^- ions combine to the protonated basic amino-acid residues with electrostatic energy. The basic amino-acid residues such as: Lys, His, Arg and amino-group of N-terminal. The opposite charges at albumin molecules surface are neutralized as the binding of I ^-, and the protective water layer is destroyed as the distribution of I^- ions between the two phases. As a result the macromolecules of HSA or BSA most possibly trend to aggregating and forming colloid in big size, so the intensity of RLS is enhanced considerably.
    The binding of I^- to HSA and BSA appropriate to the phase distribution model and related physiological effect need further studies.

REFERENCES
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