Bioconjugate Chem. 2007, 18, 2109–2114
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Intermolecular Interaction of Avidin and PEGylated Biotin Shan Ke,†,‡ John C. Wright,† and Glen S. Kwon*,‡ Department of Chemistry, University of Wisconsin—Madison, 1101 University Avenue, Madison, Wisconsin 53706-1322, and Division of Pharmaceutical Sciences, School of Pharmacy, University of Wisconsin—Madison, 777 Highland Avenue, Madison, Wisconsin 53705-2222. Received June 6, 2007; Revised Manuscript Received July 19, 2007
The equilibrium binding constants and stoichiometries between PEGylated biotins and avidin have been studied for a range of PEGylated biotin molecular weights. These studies show that as the molecular weight of PEG (polyethylene glycol) increases over the range 588, 3400, and 5000 g/mol, the equilibrium dissociation constants of PEGylated biotins with avidin increase to ∼10-8 M compared with 10-15 M for the biotin–avidin complex. The stoichiometries of PEGylated biotins with avidin are 4:1 for 588 and 3400 g/mol PEG and 1:1 for 5000 g/mol PEG. The data demonstrate that the equilibrium binding constant and the stoichiometry of the avidin–biotin–PEG complex system can be adjusted by the length of PEG chains. This approach may be used with PEGylated biotin analogues for pretargeting in drug delivery, such as a biotin-PEGylated enzyme for converting an inactive prodrug into a cytotoxin. When a PEG chain is chosen as an appropriate spacer, the length of the PEG chain must be considered because PEG can block the binding sites on avidin.
INTRODUCTION We have attempted to modify therapeutic proteins with PEG (polyethylene glycol) and deliver the PEGylated proteins to tumor tissues through the enhanced permeability and retention (EPR) effect (1). The EPR effect results in the selective sequestration of the therapeutic proteins in tumors. After administration of prodrugs, the therapeutic proteins can convert the prodrugs into toxic forms that kill tumor cells. The efficacy of this process depends on optimizing the delivery of the therapeutic proteins to the tumor while eliminating the proteins from the plasma before administration of the prodrug. The enhancement of the plasma half-life of a PEGylated protein is an important prerequisite for EPR targeting tumor tissues and the size of a PEGylated protein is a crucial factor to keep the PEGylated protein circulating in the plasma. It is generally recommended that the size of a therapeutic protein should be larger than the glomerular filtration threshold in order to avoid being cleared too quickly by the renal system (2). We believe that an ideal avidin–biotin–PEG–enzyme complex has a molecular weight higher than the glomerular filtration threshold and can increase the EPR tumor targeting. At the same time, this complex must dissociate into avidin and biotin–PEG–CPA (CPA, carboxypeptidase A) in the circulation system so that it can be cleared by the renal system before administration of the prodrug. Once the glomerular filtration threshold is reached, the half-life of the avidin–biotin–PEG–CPA complex in the plasma is primarily dependent on the dissociation kinetics of the complex that allows it to be removed by the renal system. The half-life of the avidin–biotin–PEG–CPA complex must be sufficiently long for significant tumor accumulation but short enough to allow the prodrug administration. The avidin–biotin technology has been an essential tool in modern bioscience (3). The unusually strong interaction of avidin and biotin is often the basis for pretargeting strategies in cancer treatments. For example, avidin or streptavidin has * To whom correspondence should be addressed. Telephone: 608265-5183. Fax: 608-262-5345. E-mail:
[email protected]. † Department of Chemistry. ‡ School of Pharmacy.
been employed as a bridge between a biotinylated target protein and a biotinyl anticancer reagent (4–7). However, the binding between avidin and biotin is so strong that the unmodified avidin–biotin system has a dissociation half-life of ∼200 days, a period that is too long to have any potential for our enzyme delivering strategy. The avidin–biotin system has to be modified to reduce the affinity. For this goal we chose biotinylated PEGs, which not only form a complex with avidin with a reduced affinity but also protects the enzyme during delivery (8, 9). Before the synthesis and characterization of the complete avidin–biotin–PEG–enzyme complex, this study examines the intermolecular interaction between avidin and PEGylated biotin as a function of the PEG molecular weight. The work focuses on three areas. (1) One area is affinity. How does the length of PEG chain affect the affinity of biotinylated PEG for avidin? (2) Another area is stoichiometry. Avidin has four binding sites for biotin with a stoichiometry of 1:4 of avidin/biotin. It was reported that PEGylated biotin has a lower affinity for avidin than biotin (10), but it is important to learn whether a PEG chain blocks the biotin terminus from binding with avidin/ streptavidin and reduces the effective stoichiometry of the complex. (3) Does a long PEG chain envelop avidin in the complex or is the PEGylated biotin preferentially excluded from the avidin?
EXPERIMENTAL PROCEDURES Materials. The reagents used in this study were commercially available analytical or higher grade materials except for PEGylated biotins, which have a purity ranging from 80% to 98%. 2-Iminobiotin and D-biotin were obtained from Sigma (St. Louis, MO). Avidin and Immuno Pure 2-(4′-hydroxyazobenzene)benzoic acid (HABA) were purchased from Pierce (Rockford, IL). Biotin–PEG(3400) and biotin–PEG(5000) were obtained from Nektar (Huntsville, AL). Biotin–PEG(588) was purchased from Quanta BioDesign (Powell, OH). 2,6-Anilinonaphthalene sulfonate (2,6-ANS) was obtained from Molecular Probes (Carlsbad, CA). Buffer. The PBS buffer was prepared with 8.00 g of NaCl, 0.20 g of KCl, 2.68 g of Na2HPO4 · 7H2O and 0.24 g of KH2PO4, diluted to 1 L with Millipore water, pH 7.4. If necessary, the pH was adjusted using NaOH or HCl. The stability of the
10.1021/bc700204k CCC: $37.00 2007 American Chemical Society Published on Web 10/19/2007
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avidin–biotin complex is insensitive to the ionic strength unless a very low ionic strength solution (
