The binding characteristics of vitamin B1 (VB1) and vitamin B9 (VB9) with trypsin were investigated by fluorescence spectrometry and UV/vis spectrophotometry under simulated physiological conditions. With the addition...The binding characteristics of vitamin B1 (VB1) and vitamin B9 (VB9) with trypsin were investigated by fluorescence spectrometry and UV/vis spectrophotometry under simulated physiological conditions. With the addition of VB1 or VB9, the intrinsic fluorescence emission intensity of trypsin was quenched by the nonradiative energy transfer mechanism. The fluorescence quenching process of trypsin may be mainly governed by a static quenching mechanism. The binding parameters such as the binding constants and the number of binding sites can be evaluated by fluorescence quenching experiments. The numbers of the apparent binding constant Kb of VB1-trypsin at different temperatures were 0.4948 and 4.8340 × 104 L/mol and the numbers of binding sites n were 0.9359 and 1.1820. Similarly, the numbers of the apparent binding constant Kb of VB9-trypsin at different temperatures were 5.9310 and 13.040 × 104 L/mol and the numbers of binding sites n were 0.9908 and 1.0750. The thermodynamic parameters, with a negative value of ΔG, revealed that the bindings are spontaneous processes and the positive values for both enthalpy change (ΔH) and entropy change (ΔS) indicate that the binding powers of VB1 and VB9 with trypsin are mainly hydrophobic interactions. And synchronous spectrums were used to study the conformational change of trypsin. In addition, the binding distances of VB1-trypsin and VB9-trypsin were estimated to be 0.55 nm and 0.87 nm according to the Förster’s resonance energy transfer theory.展开更多
The interaction between captopril, an inhibitor of angiotensin converting enzyme and human serum albumin, a principal plasma protein in the liver has been investigated in vitro under a simulated physiological conditio...The interaction between captopril, an inhibitor of angiotensin converting enzyme and human serum albumin, a principal plasma protein in the liver has been investigated in vitro under a simulated physiological condition by UV-vis spectrophotometry and fluorescence spectrometry. The intrinsic fluorescence intensity of human serum albumin was strongly quenched by captopril. The binding constants and the number of binding sites can be calculated from the data obtained from fluorescence quenching experiments. The negative value of ΔG0 reveals that the binding process is a spontaneous process. According to the van’t Hoff equation, the standard enthalpy change (ΔH0) and standard entropy change (ΔS0) for the reaction were calculated to be 35.98 KJ●mol-1 and 221.25 J●mol-1 K. It indicated that the hydrophobic interactions play a main role in the binding of captopril to human serum albumin. In addition, the distance between captopril (acceptor) and tryptophan residues of human serum albumin (donor) was estimated to be 1.05 nm according to the F?rster’s resonance energy transfer theory. The results obtained herein will be of biological significance in pharmacology and clinical medicine.展开更多
文摘The binding characteristics of vitamin B1 (VB1) and vitamin B9 (VB9) with trypsin were investigated by fluorescence spectrometry and UV/vis spectrophotometry under simulated physiological conditions. With the addition of VB1 or VB9, the intrinsic fluorescence emission intensity of trypsin was quenched by the nonradiative energy transfer mechanism. The fluorescence quenching process of trypsin may be mainly governed by a static quenching mechanism. The binding parameters such as the binding constants and the number of binding sites can be evaluated by fluorescence quenching experiments. The numbers of the apparent binding constant Kb of VB1-trypsin at different temperatures were 0.4948 and 4.8340 × 104 L/mol and the numbers of binding sites n were 0.9359 and 1.1820. Similarly, the numbers of the apparent binding constant Kb of VB9-trypsin at different temperatures were 5.9310 and 13.040 × 104 L/mol and the numbers of binding sites n were 0.9908 and 1.0750. The thermodynamic parameters, with a negative value of ΔG, revealed that the bindings are spontaneous processes and the positive values for both enthalpy change (ΔH) and entropy change (ΔS) indicate that the binding powers of VB1 and VB9 with trypsin are mainly hydrophobic interactions. And synchronous spectrums were used to study the conformational change of trypsin. In addition, the binding distances of VB1-trypsin and VB9-trypsin were estimated to be 0.55 nm and 0.87 nm according to the Förster’s resonance energy transfer theory.
基金the National Key Technology R&D Program of China(No.2008BAJ08B13)for financially supporting this work.
文摘The interaction between captopril, an inhibitor of angiotensin converting enzyme and human serum albumin, a principal plasma protein in the liver has been investigated in vitro under a simulated physiological condition by UV-vis spectrophotometry and fluorescence spectrometry. The intrinsic fluorescence intensity of human serum albumin was strongly quenched by captopril. The binding constants and the number of binding sites can be calculated from the data obtained from fluorescence quenching experiments. The negative value of ΔG0 reveals that the binding process is a spontaneous process. According to the van’t Hoff equation, the standard enthalpy change (ΔH0) and standard entropy change (ΔS0) for the reaction were calculated to be 35.98 KJ●mol-1 and 221.25 J●mol-1 K. It indicated that the hydrophobic interactions play a main role in the binding of captopril to human serum albumin. In addition, the distance between captopril (acceptor) and tryptophan residues of human serum albumin (donor) was estimated to be 1.05 nm according to the F?rster’s resonance energy transfer theory. The results obtained herein will be of biological significance in pharmacology and clinical medicine.
