Hu Qiuluan ^a, Wang Chunfeng^b, Li Quanmin^c
（^aDeparment of Chemistry, Luoyang Normal University, Luoyang ,471022, China; ^bCollege of life Science, Henan Normal University, Xinxiang, Henan, 453007, China; ^cCollege of Chemistry and Environmental Science, Henan Normal University, Xinxiang, Henan, 453007, China）

Received on Jan. 31, 2006; Supported by the Natural Science of Henan's Fundation (0411021600).

1. INTRODUCTION
In the past years, the quantification of protein in biological samples has drawn great attention as life science and related research activities move along. Methods for separation and analyzing proteins are booming up to a great extent, of which the spectrophotometry has been considerably recognized because it is simple, fast, cost-effective and precise^[1-3]. At present, the study for spectrophotometric determination of proteins mainly concentrates on small molecule such as organic dyes as spectral probe, and there is less report on the research of the metal complex as spectral probe. Recent researches indicate that the determination of protein concentration by using metal complex as spectral probe can obtain higher sensitivity and selectivity ^[4-6]. In this paper, the authors for the first time report the binding interaction of protein with Cu (II)-Nitrosulfophenol S Complex , and designed a new method for the determination of proteins by using the metal complex as spectral probe. The sensitivity of this method is higher than most of the spectrophotometric methods which the dyes are directly used as spectral probe. Many inorganic ions and organic materials do not interfere with the determination. The method has been applied to the determination of total amounts of proteins in the human serum samples and satisfactory results are obtained.

2. EXPERIMENTAL
2.1 Reagents
Nitrosulfophenol S (Abbr. R in this paper) was obtained from Beijing chemical plant (5.0×10^-4 mol/L). Bovine serum albumin (BSA), human serum albumin (HSA), g-globulin (g-G), hemoglobin (Hb), egg albumin (OVA), lysozyme (Lys) were purchased from Sigma. 1.0×10^-3mol/L Cu(NO₃)₂ solution; 0.1mol/L NaCl solution.1g/L glucose solution; 0.5 g/L Citric acid; 1％（V/V）TritonX-100 solution; 100mg/L tetradecylpyridinium bromide(TPB) solution; 100mg/L Sodium dodecyl sulfonate (SDS) solution; ethanol; pH1.8-2.2 Clark-lubs (C-L) buffer solution was prepared by dissolving some KHC₈H₄O₄ and some HCl in distilled water and diluting it to some volume. It was used to control pH values of the test solution. All of the reagents were of analytical grade, doubly-deionized water used throughout.
2.2 Apparatus
GBC UV/VIS 916 spectrophotometer (GBC Company, Australia) was used for recording the absorption spectra, and a Model 723 spectrophotometer (Shanghai Jingmi Yiqi Ltd, China) for the measurement of absorbance at a given wavelength. PHS-3B pH meter (Shanghai Leici) was used to determine pH values.
2.3 Procedure
The following solutions were added to a 10mL tube:1.5mL C-L buffer solution (pH2.0),1.0mL Nitrosulfophenol S solution(5.0×10^-4mol/L),1.0mL Cu(II) solution(1.0×10^-3mol/L )and HSA standard solution. The mixture was diluted to 10mL with water. After 10min, the absorbance (A₀) of Cu (II)-Nitrosulfophenol S complex solution and the absorbance (A) of Cu (II)-Nitrosulfophenol S-HSA solution were measured at l=617nm using water as the blank. DA(A₀-A) was calculated.

Fig.1 Absorption spectra of Nitrosulfophenol S ,Cu(II)-Nitrosulfophenol S and Cu(II)-Nitrosulfophenol S-HSA
1 Nitrosulfophenol S (C_R=5.0×10^-5mol/L )against water; 2. Cu (II) -Nitrosulfophenol S-HSA (C_Cu-R=5.0×10^-5mol/L ) against water
C_HSA (mg/L): a. 0; b. 20; c. 40; d. 60; e. 80

3. RESULTS AND DISCUSSION
3.1 Absorption spectra and reaction mechanism
In the pH2.0 C-L buffer solution, Cu (II)-Nitrosulfophenol S complex solution is blue. The absorption spectra of Cu (II)-Nitrosulfophenol S complex are shown in Fig.1. 1, with three absorption peaks, at 495nm, 617nm and 658nm, respectively. After HSA is added, the fading in color is observed. Two peak values at 617nm and 658nm obviously decrease, and the decrease of absorbance is proportional to the concentration of proteins (shown in Fig. 1). Based on the above observation, a new method for the spectrophotometric determination of proteins concentration is established. As the change of absorbance at 617nm is larger than that of absorbance at 658nm, the 617nm is chosen as the determination wavelength for Cu (II)-Nitrosulfophenol S-staining protein in the experiment.
    Three surfactants were investigated, respectively. The experimental results indicate that SDS and TritonX-100 do not react with Cu (II)-Nitrosulfophenol S complex, while TPB make absorbance decrease (shown Fig. 2). Fig.1 and Fig.2 show that the binding interaction of HSA with Cu (II)-Nitrosulfophenol S complex is similar to the binding interaction of cation surfactant (TPB) with Cu (II)-Nitrosulfophenol S complex. In acidic medium at pH2.0, Cu (II)-Nitrosulfophenol S complex have negative charge because of the ionization of sulfonic acid groups . In the pH range of 1-4, the proteins have positive charges and molecules present high extension state ^[7]. Therefore Cu (II)-Nitrosulfophenol S complex reacts with proteins to form a stable ternary compound (Cu (II)-Nitrosulfophenol S-HSA) by electrostatic attraction, which is similar to the binding interaction of cation surfactant with the complex ^[8,9].

Fig.2 Absorption spectra of Cu (II)-Nitrosulfophenol S and copper(II)-Nitrosulfophenol S-TPB
a-d. Cu(II) -Nitrosulfophenol S-TPB against water
C_TPB (mg/mL): a. 0; b. 10; c. 20; d. 40

3.2 Effect of pH on the reaction　
The effect of the different pH values on the fading reaction was studied. The result implies that DA of the system reaches a plateau and kept constant in the pH range of 1.8-2.2 and in the dose of buffer solution range of 1.0-2.0mL. Therefore, 1.5mL buffer solution (pH2.0) was chosen to maintain pH values of the test solutions.
3.3 Effect of Cu(II) and Nitrosulfophenol S concentration
Cu (II) can react with Nitrosulfophenol S to form 1:1 complex^[10] in acidic medium. In the course of forming complex, it is generally needed to add excessive ligand or the center ion. When proteins react with Cu (II)-Nitrosulfophenol S complex, proteins mainly combine with sulfonic acid group of Nitrosulfophenol S by electrostatic attraction. Therefore, in order to receive stable Cu (II)-Nitrosulfophenol S complex, the dose of Cu (II) should be increased. The experimental result shows that DA of the system reaches its maximum and keeps constant when both Cu (II) solution(1.0×10^-3mol/L) and Nitrosulfophenol S(5.0×10^-4mol/L) solution are at the range of 0.8-1.2mL. So1.0mL Cu (II) solution and 1.0mL Nitrosulfophenol S solution were chosen in the experiment.
3.4 Reaction time, temperature and stability of complex
The effect of reaction time was tested at 20°C (room temperature), 25°C and 35°C (water bath), respectively. Results demonstrate that : (1) the reaction between Cu (II)-Nitrosulfophenol S complex and HSA occurs rapidly at the above temperatures(<10min).(2) DA is kept constant within 1 hour. (3) the temperatures have no effect on the reactive system.
3.5 Effect of ion strength and organic solvent
The effect of ion strength on the reactive system was tested with NaCl solution (0.1 mol/L). As Fig. 3 shows, the absorbance difference of the system is constant in the concentration of NaCl range of 0-0.02 mol/L. The effect of ethanol on the reaction was studied. When ethanol is in the range of 0-20%(V/V), both the absorbance of Cu (II)-Nitrosulfophenol S complex (against water) and the absorbance of Cu (II)-Nitrosulfophenol S complex-HSA (against water) increase. However, the increased degree is not the same, therefore ethanol has some effect on the system as Fig. 3 shown.
3.6 Effect of surfactants
In order to understand whether surfactants could increase sensitivity of the system, three surfactants were tried out, respectively. The results are shown in Fig. 3. DA of the system has not been affected by the neutral TritonX-100. TPB makes DA of the system increased. The reason may be that TPB can react with Cu (II)-Nitrosulfophenol S complex to form a ternary complex. TPB participates in the fading reaction, which makes DA of the system increased. On the contrary, SDS makes DA of the system reduced. As SDS (anion surfactant) are bound to proteins, the surface of the SDS-protein complex ^[11] will be negatively charged, which greatly affects the binding interaction proteins with Cu (II)-Nitrosulfophenol S complex because of electrostatic repulsion. At the same time, it is found that the absorbance of Cu (II)-Nitrosulfophenol S complex(against water) was hardly affected by SDS. Therefore, DA of the system reduced.

Fig.3 The effect of ion strength, organic solvent and surfactants
(C_R=5.0×10^-5mol/L ; C_BSA=7.35×10^-7mol/L )
1% (V/V) TritonX-100 solution; 100mg/L TPB solution; 100mg/L SDS solution; 0.1 mol/L NaCl solution

3.7 Precision of the assay
Ten standard HSA solutions were measured, where each concentration was kept at 7.35×10^-7mol/L, the average absorbance of system was 0.313 at l=617nm, RSD was 2.9%.
3.8 Calibration curves
Calibration curves, linear range, apparent molar absorptivity (e) and Sandell's sensitivity (S) of BSA, HSA, Hb, g-G, OVA, Lys were obtained under the optional condition. All the analytical parameters are listed in Table 1. Obviously, this assay has the following characteristics: wide linear range, high sensitivity and good response on many proteins.

Table 1 Standard curves for some proteins