Serum albumin (SA) is one of the most abundant proteins in the blood circulation, and has important physiological functions such as storage, transportation and organism protection. Bovine serum albumin (BSA) has been used as an ideal protein model for the study of physicochemical properties, structure and function due to the relatively small molecular weight and solubility. 8-anilino-naphthalene-1-sulfonic acid (ANS) and its ammonium salts are intermediates of various azo dyes and are commonly used as fluorescent probes. In this paper, we will introduce the interaction between ANS and BSA, and conformational changes of BSA in the fluorescence method with ANS as probe.

Interaction of ANS and BSA

As a hydrophobic fluorescent probe, ANS (or ANS-NH4) is widely used to study the conformational changes of BSA. The interaction between the two is mainly electrostatic attraction [1], ANS and BSA have a strong binding site, where electrostatic interaction play an important role. The presence of sulfonic acid groups played a huge role. Sulfonic acid molecules are water-soluble, have a large polarity of the surface and molecular flexibility, and can act with charged groups of protein by electrostatic attraction. Because of greater molecular flexibility, it is more suitable for assembly of protein hydrophobic area. And the sulfonic acid group enhance the water-soluble of ANS, and contact with BSA more easily. At the same time, the increase in the polarity of the surface of the ANS is less likely to penetrate the lipid biofilm, reducing its risk of entering the biological body.

Changes of BSA in solution

With ANS as the fluorescent probe, the change of endogenous fluorescence of hydrophobic tryptophan residue and its microenvironment was studied to detect the conformational change of BSA in solution.

Guanidine hydrochloride solution

Urea solution

Compared with guanidine hydrochloride, BSA is more stable in urea [3]. The changes of BSA in urea and the critical concentration of conformational change were described according to the change of fluorescence parameters. The results showed that the conformational changes of BSA also included the change of tryptophan residue and the binding region of ANS probe in the process of urea denaturation with the increase of urea concentration: The probe-binding region is more sensitive to urea, and the low-concentration denaturant can cause its conformational changes to become loose and expose to the hydrophilic solvent. The tryptophan residue region is resistant to low concentrations of urea (0~1.0mol•L-1), the denaturation process undergoes a triaxial process with contraction-extension, and its conformational change rate reaches a maximum at a concentration of 3.0~5.0mol•L-1, and critical concentration at 5.4mol•L-1, the tryptophan residue has been completely exposed to the solvent at a concentration of 7.8mol•L-1 and in an irreversible depolarized state.

To understand the interaction between ANS and BSA, and study the change of protein conformation from the molecular level, establish the law of protein conformation with the change of environment, could provide more relevant information and theory for the research of protein and the progress of biological genetics and molecular medicine.

References

[3] Zhang Jiaxing, Jin Ke, Yin Zongning. Characterize the denaturation process of bovine serum albumin in urea by fluorescence parameters. Huaxi Pharmaceutical Journal, 2012,27, 371 ~ 375.