Cysteine-Free Mutant of Aequorin as a Photolabel in Immunoassay Development Suresh Shrestha, † Insook R. Paeng, ‡ Sapna K. Deo, † and Sylvia Daunert* ,† Departments of Chemistry and Pharmaceutical Sciences, University of Kentucky, Lexington, Kentucky 40506-0055 and Department of Chemistry, Seoul Women’s University, Seoul, Korea. Received April 5, 2001; Revised Manuscript Received September 27, 2001 The bioluminescent protein aequorin is a sensitive label that has been employed in a number of analytical applications. A mutant of aequorin with enhanced stability produced recombinantly in our laboratory has been employed as a label in the development of an immunoassay for digoxin. Digoxin is a cardiac glycoside used in the treatment of congestive heart failure. This drug has a very narrow therapeutic range of 0.8-2.0 ng/mL (1.0-2.5 nmol/L), thus requiring therapeutic drug monitoring. In this study, a derivative of digoxigenin was chemically conjugated to the mutant aequorin, and the resulting protein-digoxigenin derivative conjugates were characterized in terms of their luminescence properties. A solid-phase immunoassay for digoxin was then developed. The detection limit of the assay for digoxin was 1 × 10 -12 M. To demonstrate the use of this mutant aequorin as a label in biological sample analysis without any need for pretreatment of the samples, the assay was tested in serum spiked with digoxin. Interference from digoxin analogues was also evaluated to determine the specificity of the assay. INTRODUCTION Aequorin, a calcium-binding photoprotein, was origi- nally isolated from the jellyfish Aequorea victoria in Friday Harbor, Washington (1). The holoprotein consists of apoaequorin (189 amino acid residues), a chromophore (coelenterazine), and molecular oxygen (2). The photo- protein is activated by calcium, which binds to the three EF-hand structures present within the protein molecule (3). These EF-hand structures are characteristic of the calcium-binding protein family. Upon addition of calcium, aequorin undergoes a conformational change leading to oxidation of bound coelenterazine to coelenteramide with the subsequent release of CO 2 and a flash of light (λ max ) 469 nm) (4). Aequorin has been employed in a variety of analytical and biological applications. It has been used in the measurement of calcium in various intracellular com- partments (5). Highly sensitive binding assays and immunoassays have been developed for peptidic and nonpeptidic analytes using aequorin as a label (6, 7). A proteolytic bond cleavage assay has also been developed using an aequorin fusion protein (8). Since aequorin can be detected down to attomole levels, it has been employed in the determination of biotin in single cells and in picoliter-volume vials (9-11). In addition, aequorin- labeled probes have been used as an alternative to radiolabels in nucleic acid hybridization assays (12, 13). Several mutational studies have been performed on aequorin to elucidate the effects of various amino acids on the activity of the protein (14-16). Among them, cysteine-free mutants have shown to increase the pro- tein’s luminescence activity. The luminescence activity of the mutant was found to be higher (124%) when compared to that of native protein (100%) (17, 18). Although the specific cause for the higher activity is not yet clear, it has been suggested that the cysteines present in the native protein are not essential in the generation of the luminescence activity (19). We have postulated that presence of disulfide bonds in the native protein may introduce conformational constraints in the protein struc- ture leading to a reduction in the bioluminescence activity (19). In addition to an increased activity, the cysteine-free mutant does not need the addition of a reducing agent to prevent dimerization of the protein, thus increasing the stability of the protein. The goal of this study was to investigate the perfor- mance of this cysteine-free mutant of aequorin con- structed previously in our laboratory as a label in immunoassay development. Bioluminescence is a rela- tively rare phenomenon in nature; thus, when aequorin is used as a label in biological sample analysis it offers the advantage of producing a low background signal (3). Therefore, we decided to investigate the detection ability of this mutant of aequorin in the analysis of biological samples and without performing any pretreatment steps. Digoxin was selected as a model analyte for the study. Digoxin is a cardiac glycoside used in the treatment of congestive heart failure and supraventricular arrhyth- mias (20, 21). It has a narrow therapeutic window of 0.8- 2.0 ng/mL with a potential of fatal toxicity. Hence, it frequently needs therapeutic drug monitoring (TDM) (22). For the purpose of this study, a derivative of digoxi- genin was employed for its chemical conjugation to the lysine residues of the mutant aequorin. Several mutant aequorin-digoxigenin derivative conjugates with various mole ratios of digoxin to aequorin were prepared and were characterized in terms of their luminescence prop- erties and binding ability to an anti-digoxin antibody. Dose-response curves were generated for digoxin in a sequential binding format. The optimized assay was used * Corresponding author: Sylvia Daunert; Phone: (859) 257- 7060; Fax: (859) 323-1069; E-mail: daunert@pop.uky.edu. † University of Kentucky. ‡ Seoul Women’s University. 269 Bioconjugate Chem. 2002, 13, 269-275 10.1021/bc010044c CCC: $22.00 © 2002 American Chemical Society Published on Web 02/21/2002