关键词: 毛细管电泳, 理论模型, 结合参数, 苦参碱, 血清白蛋白

Abstract:

Matrine (MT) is an alkaloid widely used in the treatment of tumor diseases. It is the main medicinal ingredient in the dried roots of kuh-seng (Sophora flavescens Ait). However, there have been few studies on its transport mechanism. Serum albumin (SA) is the most abundant protein in blood. SA combines easily with many substances, including MT. MT and human serum albumin (HSA) were analyzed by capillary electrophoresis (CE) under in vitro conditions. The capillary tubing was 50 μm. The total length of the capillary was 60 cm, the total effective length was 50 cm. The interaction models of ligand-receptor binding were constructed by the mobility and frontal analysis (FA) methods. The purpose of establishing the interaction model was to study the binding of MT and SA. The phosphate buffer solution (PBS, 0.02 mol/L) was prepared in double distilled water. All solutions were prepared in PBS (0.02 mol/L). All solutions were filtered twice through a 0.45 μm microporous membrane, degassed for 5 min at a time. In the mobility method, different gradient MT solutions were used as running buffers. Their concentrations were 1.0×10^-4-1.0×10^-3 mol/L, with the gradient of 1.0×10^-4 mol/L. And the HSA solution containing (0.5% (v/v)) acetone was used as test sample. Its concentration was 1.0×10^-5 mol/L. The nonlinear fitting method was used to obtain the binding parameters of MT and HSA. In the FA method, different gradient MT-HSA solutions were used as test samples. Their concentrations were 1.0×10^-4-1.0×10^-3 mol/L, with the gradient of 1.0×10^-4 mol/L. And the PBS solution (0.02 mol/L) was used as running buffer. Then three equations were used to obtain the binding parameters of MT and HSA. And the applicability of the models was analyzed using the binding parameters. These three equations were nonlinear regression equation, Scatchard linear equation, and Klotz linear equation. Using the mobility method, the apparent binding constant K_B was 8.072×10³ mol/L. According to the FA method, three apparent binding constants were obtained for MT and HSA. The apparent binding constant K_B of HSA and MT by nonlinear regression equation, Scatchard linear equation and Klotz linear equation were 1.434×10³, 1.781×10³ and 2.133×10³ mol/L. The comparison was as follows, K_{B(nonlinear regression equation)} < K_{B(Scatchard linear equation)} < K_{B(Klotz linear equation)}. The number of binding sites was about 1.0. It was indicating that MT had only a single type of binding site with HSA. By analyzing the applicability of the model, the correlation coefficients (r) of the three equations were obtained. The comparison was as follows, r_{(Klotz linear equations)} > r_{(nonlinear regression equations)} > r_{(Scatchard linear equations)}. The results showed that both the methods were all suitable for analyzing the MT-SA system. The FA method could calculate the apparent binding constants and the numbers of binding sites. Therefore, it was more suitable for the analysis of MT and HSA. And the Klotz linear equation was the best fit for the theoretical model among the three equations. The combined parameters indicated that the interaction of MT with HSA had only one binding site. And the binding of MT with HSA was stable. This experimental method could be used to determine the binding status of MT and HSA. It is useful to further explore the binding mechanism of MT and HSA. This work provides valuable information on the interaction mechanism of typical alkaloids with SA. It will be useful in studies of the blood transport mechanisms of alkaloids.

Key words: capillary electrophoresis (CE), theoretical model, combined parameters, matrine (MT), serum albumin (SA)