Study on the Interaction between the chiral drug of propranolol and α1-acid glycoprotein by fluorescence spectrophotometry

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Abstract

The interaction between the chiral drug of propranolol (PPL) and α1-acid glycoprotein (AGP, orosomucoid) has been first studied by fluorescence spectrophotometry. The fluorescence intensity of PPL increased due to the addition of AGP into PPL. The equation of Scatchard was employed to calculate the association constant and binding site number of the two enantiomers with AGP. The association constant is 2.62 × 105 M−1 for R-PPL and 8.57 × 105 M−1 for S-PPL and the binding site number is 0.41 for R-PPL and 1.17 for S-PPL at 17 °C respectively. The method of thermodynamics was applied to determine the binding type of S-PPL with AGP. The results suggested that the binding type is mainly van der waals force or hydrogen bond. At last the effect of three metal cations on the association constant and the binding site number of S-PPL with AGP was examined.

Introduction

An extensive attention has been paid to study on the interaction between drug and biology macromolecule (such as protein, DNA, etc.) in the scope of life science, chemistry and clinic medicine [1]. The interaction of drug with protein in serum can influence bioavailability of drug and affect the function of several biomolecules. Clinical effect of the plasmatic level of a drug is a very important pharmacological parameter determined by absorption, distribution and elimination. Chiral drug plays an important role in our daily administration of medicine. The different isomers have different clinical effects. Probably one isomer can cure disease while the other isomer can result in side effect, even make disease worse, and maybe it is fatal [2], [3], [4].

The human α1-acid glycoprotein (AGP, orosomucoid) is a small acute-phase glycoprotein (Mr  44,000) that is negatively charged at physiological pH. It consists of a chain of 181 amino acids, and contains 40% carbohydrate by weight and has 16 sialic acid residues (10–14% by weight). Five heteropolysaccharide groups are linked via an N-glycosylic bond to asparaginyl residues of the protein. The protein contains tetrantennary as well as di- and triantennary glycans [5], [6], [7], [8]. In the acute phase (e.g. cancer or inflammation and so on), its content increases evidently. It is a very important protein in the blood, which binds to basic drugs. Propranolol (PPL, Fig. 1) is one of most widely used and well-know racemic drugs, a blocking drug for treating certain cardiovascular via restraining sympathetic nerve from undue excitement or excreting catecholamine. So it can be used as anti-arrhythmia drug. Its main carrier protein is α1-acid glycoprotein [9], [10]. PPL has two isomers, and its racemate is commonly used for treatment. But R-PPL can bring about side effect.

Presently, the methods employed to study the interaction of drug with protein mainly include: ultraviolet spectrophotometry, fluorescence spectrophotometry, chromatography (GC, LC, CE, etc.), equilibrium dialysis [5], [11], [12], [13], [14], [15] and so on. Previously, there were several methods to study the reaction mechanism of PPL with AGP, for example capillary electrophoresis [5], equilibrium dialysis [14] and so on. However, those methods were laborious and time consuming.

In this paper, fluorescence spectrophotometry was first employed to study the interaction of PPL and AGP. The results can explain why S-PPL is more effective than R-PPL. The proposed method is very rapid and highly sensitive because sample preparation and fluorescence determination are simple and facility.

Section snippets

Materials and apparatus

AGP (purity >99%), and R- and S- enantiomers of PPL were purchased from Sigma Chemical Co. F-5301PC fluorescence spectrophotometer (Shimidzu Company, Jap.), pH 25 pH meter (Shanghai, China). The other reagents were all analytical purity. Water used in the experiment was doubly distilled water.

Spectroscopic measurement

Aqueous stock solution (1.0 ml) of R-, S- or DL-PPL was accurately transferred into a 5 ml volumetric flask, and then an appropriate amount of 2.0 × 10−5 mol/L AGP solution and 1 ml of 0.05 mol/L Tris–HCl buffer

Effect of pH on the fluorescence of PPL

The effect of pH on the fluorescence of DL-PPL was first examined. The results suggested that the fluorescence intensity of PPL decreased in the hard acidic or alkaline solution. Moreover, the red shift of the maximum emission wavelength of PPL was observed in above conditions. As can be seen from Fig. 2, the fluorescence intensity of PPL decreased when pH was above 9.5 or below 2.3. But the fluorescence intensity change of PPL almost did not take place between pH 2.3 and 9.5. So pH 7.0 was

Conclusion

The fluorescence method is highly sensitive and convenient in the study of intermolecular interaction. Both of two enantiomers of PPL can bind to α1-acid glycoprotein to lead to fluorescence enhancement. But the association constant of S-PPL with AGP is about 2.3 times higher than that of R-PPL, and the binding site number of S-PPL with AGP is also more than that of R-PPL. The type of interaction between PPL and AGP is mainly hydrogen bond or van der waals force. The reaction between PPL and

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