According to the Center for Disease Control, chronic diseases such as heart disease and cancer account for over 50% of all deaths in the United States. In chronic inflammatory diseases, a group of proteins called acute phase proteins are secreted at a higher rate in the plasma of the human circulatory system. One such protein, alpha-1-acid glycoprotein (AGP), is produced at an increased level by the hepatocytes in the presence of stress-inducing stimuli and trauma, particularly with chronic inflammation. As a main drug-binding protein in human serum, this increase in concentration of AGP in the circulation will affect how the body processes drugs used for treatment. By studying how various drugs interact with AGP, valuable information can be obtained about their effectiveness. High performance affinity chromatography (HPAC) is a method in which biologically relevant ligands, such as proteins or antibodies, are immobilized to a support and used as the stationary phase in typical HPLC systems. This technique has been used regularly to determine drug-protein binding. In this study, AGP was entrapped within a microcolumn using glycogen-capped dihydrazide activated silica in order to maintain the protein's native soluble form. The interactions between AGP and S-propranolol, a drug used to treat hypertension and anxiety, were studied using this microcolumn by frontal analysis and zonal elution competition studies to obtain information on both equilibrium association constants and the number of binding sites within the column. The association constant measured for S-propranolol using frontal analysis was 1.62 (�0.87) x106 M-1, similar to a previously measured value of 1.4 (�0.6) x106 M-1. In competition studies using carbamazepine as the injected analyte, the association constant for S-propranolol measured was 1.27 (�0.18) x106 M-1. These results can be used to further understand drug-protein interactions and may improve chronic inflammatory disease treatment for people in the future.

Chromatographic approach to drug-protein binding studies using entrapped alpha-1-acid glycoprotein microcolumns

245th National Meeting (2013)

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