engleski

Separation of the native and desialylated human alpha-1-acid glycoprotein sialoforms using low pressure pH gradient ion-exchange chromatography

Alpha-1-acid glycoprotein (AGP) is an important plasma protein involved in the binding and transport of many basic and neutral drugs.[1] According to the free drug hypothesis, only the unbound drug is available to act at physiological sites of action, thus the importance of plasma protein binding primarily resides in its impact on pharmacokinetic properties such as clearance and volume of distribution.[2] The purpose of this study is to separate the native (sialylated) AGP from the desialylated AGP using low-pressure pH gradient ion-exchange chromatography which will further be used in drug-binding studies of different AGP sialoforms. Desialylated AGP is prepared by incubation of native AGP in the Immobilized SialEXO Microspin columns (Genovic AB, Sweden) with end-over-end mixing at room temperature. Sialoform separation is performed using specialized pH gradient chromatography buffers (pIsep, CryoBioPhysica Inc., USA). The mixture of both native and desialylated AGP in buffer A (pH = 6) is injected onto HiTrap Q HP 1 mL anion exchange chromatography column (GE Healthcare Bio-Science AB, Sweden). Elution is done by single-step linear gradient (0-100 % pIsep B, pH = 2) using ÄKTA Start FPLC system (GE Healthcare, USA). Protein quantity in the eluate is monitored by absorbance measurement at 280 nm. Afterward, the pH of each fraction is measured. The obtained pH values are used for the pI value approximation of the native and desialylated AGP. The observed pI values of sialoforms differ significantly and hence can be fully separated. This method provides quick, simple, and cost-effective AGP sialic acid content testing, and can be easily modified for other glycoproteins. REFERENCES [1] Z. Huang, T. Ung, Current Drug Metabolism 2013, 14, 226-238. [2] S. A. Smith, N. J. Waters, Pharmaceutical Research 2019, 36, 30.

Alpha-1-acid glycoprotein ; Sialoform ; Separation

nije evidentirano