Interaction Of Bile Acids And Protein Stability And Class Composition

Objective:Clarify the stability of BAs and the interaction between BAs and BSA, which did an overview for the quality of QKL and clinical reference for BAs. Methods:The contents of CA and HDCA in the intermediates (mixed solution of CA and HDCA, mixed solution of four ingredients, mixed solution of six ingredients, mixed solution of eight ingredients) and10batches of QKL were assayed respectively. The paper also explored the factors that affected the stability of CA and HDCA raw materials on the basis of the determination of CA and HDCA in QKL injection by comparing the content change on the0day,5th day,10th day of CA and HDCA raw materials under the condition of high temperature (40℃and60℃), light (45001x±5001x), humidity (relative humidity75%±5%and90%±5%) and the content change on0day,5th day,10th day of CA and HDCA in QKL injection under light (45001x±5001x) and high temperature (40℃and60℃) factors. Based on the stability study of CA, HDCA raw materials and QKL injection, we used LC-MS technique to further study about the product of interconversion. To futher study the nature of CAand HDCA, the interaction be-tween six micromolecules of BAs (CA、CA、DCA、HDCA、UDCA、CDCA、TCDCA) and BSA was investigated by FS combined with FT-ATR-IR under the conditions of ionic strength0.15mol·L-1and pH7.40which own to simulated physiological status. According to the dynamic quenching rate constants Kq of Stern-Volmer equation, we could infer the fluo-rescence quenching mechanism of BSA; The binding constants KLB and every type of conju-gation effort of these compounds and BSA were calculated respectively according to Lin-eweaver-Burk equation and thermodynamic parameters; Besides, the binding site numbers n and binding constants KA in terms of Langmuir equation were obtained aswell; The effects of BAs on the conformation of BSA were also researched by synchronous fluorescence tech-nique. Meanwhile, the paper also used docking technology to simulate binding state between six BAs with BSA to show the possible interaction sites. To study the protein binding rate of BAs including CA and HDCA with BSA, HPLC was employed to determine the concentra-tion of BAs in the PBS. The protein binding rates were studied by balance dialysis. And ac-cording to the concentration ratio of inside and outside the dialysis bag determined at different time to ensure the balance time of free diffusion. Result:The average content of CA and HDCA were5.79.0、1.02、2.12、2.07mg·mL-1and4.59、0、0.81、1.10、0.80mg·mL-1respectively in mixed solution of CA and HDCA, mixed solution of four ingredients, mixed solution of six ingredients, mixed solution of eight ingredients and QKL. The study of influ-encing factors on the stability for BAs raw materials showed that the contents of both gradu-ally decreased; What is more, the content of HDCA decreased more significantly compared with the content of CA. And the content change of CA in QKL injection was not obvious, but there were certain decline on the10th day of HDCA. However the content change of CA and HDCA in QKL injection was more stable than CA and HDCA raw materials. CA had converted to DCA partially, while HDCA had partially converted into its isomer CDCA and another component was to be studied by LC-MS technology. The experimental results of spectrometry indicated that, BAs could insert into the BSA molecule initiatively to become ground-state complex sub-stances which resulted into intrinsic fluorescence quenching. The quenching mechanism was mainly hydrogen bonding or Vander Walls force, and the influence on the framework of BSA was not obvious. At the same time, we studied the effects of BAs on the conformation of BSA by FT-ATR-IR. The results showed that the relative intensity of amide Ⅰ and amide Ⅱ band changed, but the peak position and peak width did not change significantly which further val-idated the less impact of BAs on the structure of BSA.By molecular docking technology, we could inferd that CA, UDCA, and TCDCA combined with BSA mainly locating at the domain Ⅱ, while CDCA were mainly locating at the domain Ⅰ of BSA. DCA formed hydrogen bonds with the amino acids located at domain Ⅰ and Ⅲ, while HDCA formed hydrogen bonds with the amino acids located at domain Ⅱ and Ⅲ of BSA. The results of bind ing rate showed that when both of the concentration of CA and HDCA were2.02,1.01,0.505(mg·mL-1) re-spectly, the binding ratio between CA and BSA was78.7%,78.2%,59.4%, while the binding ratio between HDCA and BSA was68.9%,76.2%,81.8%respectively. Conclusion:The compatibility relations and pH value of QKL injection were conducive to the stability of CA and HDCA ingredient. There was certain binding interaction between BAs and BSA which provided the condition for storage and transport of protein in the body. There was moderate intensive binding rate between CA/HDCA and BSA, so there was no side effect due to free over-dose of CA compounds in vivo. At the same time, the interactional mechanism was clar-ified, and the theoretical basis was provided for the further study of the inner mechanism and biological effect in organism of BAs and the protein.