392 ISSN 0326-2383 KEY WORDS: All-trans retinoic acid (ATRA), 4-amino-2-trifluoromethyl-phenyl retinate (ATPR), fluorescence spectroscopy, Gel filtration, Plasma protein-binding rate, Quenching constant. * Author to whom correspondence should be addressed. Jianping Zhou E-mail: zhoujpcpu@126.com * Author to whom correspondence should be addressed. Feihu Chen E-mail: cfhchina@sohu.com Latin American Journal of Pharmacy (formerly Acta Farmacéutica Bonaerense) Lat. Am. J. Pharm. 32 (3): 392-9 (2013) Regular Article Received: November 6, 2012 Revised version: January 10, 2013 Accepted: January 12, 2013 Interaction of a Novel All-Trans Retinoic Acid Derivative with Bovine Serum Albumin and Human Plasma Protein Studied by Gel Filtration (Sephadex LH-20) and Fluorescence Quenching Method Jihui TANG 1,2 , Jing YAO 2,3 , Ayman Y. WADDAD 2,3 , Jianping ZHOU 2,3 * & Feihu CHEN 1 * 1 College of Pharmacy, Anhui Medical University, 81 Meishan Road, Hefei 230032, China 2 Department of Pharmaceutics, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, China 3 State Key Laboratory of Natural Medicines, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, China SUMMARY. ATPR (4-amino-2-trifluoromethyl-phenyl retinate) was derived from all-trans retinoic acid (ATRA). In order to make a primary estimation on the possible interaction between carrier proteins and ATPR or ATRA in vivo, the two drugs’ binding behaviors to bovine serum albumin (BSA) were analyzed with fluorescence quenching method, and also, their human plasma protein-binding rates were tested with a novel method of gel filtration (Sephadex LH-20). The experimental results showed that while the attrac- tion between ATPR and BSA was mainly due to electrostatic force, attractions between ATRA and BSA came from both the electrostatic and van der Waals forces. The results also confirmed that the fluores- cence quenching of the BSA-ATRA and -ATPR was mainly static quenching, with the quenching constant K a of ATRA-BSA being 30 times more than that of ATPR-BSA. In addition, both the plasma protein-bind- ing rates of ATRA and ATPR to human plasma were higher than 90%. INTRODUCTION Serum albumin is the most abundant protein in human blood plasma, about 42 g/L, account- ing for 60% of the total protein 1 . Serum albu- min plays an important role in maintaining the colloidal osmotic pressure of the blood, trans- porting and distributing exogenous and endoge- nous molecules and metabolites such as nutri- ents, hormones, fatty acids and diverse drugs 2 . As a result, when a drug circulation in blood stream, a possible reversible binding can occur between the drug and serum albumin and a fi- nal equilibrium will be reached between the bound and free molecular species. Therefore, the in vivo fate of a drug is directly influenced by its binding degree to serum albumin. And only the free drug molecule can cross mem- brane barriers and is subject to distribution, metabolism and glomerular filtration 3 . Howev- er, understanding the interaction between drugs and serum albumin can not be accomplished without investigating two important parameters: (1) association constant and (2) plasma protein binding rate. Association constant describes the binding affinity between a drug and serum albu- min at equilibrium state. Bovine serum albumin (BSA) is a model protein often used to study the interaction between drug and serum albumin due to the conformation similarity between BSA and human serum albumin (HSA), representing 76% of amino acid sequence homology. BSA consists of 583 amino acid residues, and has two tryptophan moieties (Trp135 and Trp214), locat- ed in subdomains IA and IIA, respectively 4 . Flu- orescence spectroscopy is often used for study- ing the drug-protein binding interaction due to its high sensitivity, selectivity, suitability and abundant theoretical foundation. The method can provide the information reflecting the con- formational change of proteins that can occurs in various physiological environments 5 . Plasma protein-binding rate represents the ratio of bind-