Journal of lmmunological Methods, 143 (1991) 263-272 © 1991 Elsevier Science Publishers B.V. All rights reserved 0022-1759/91/$03.50 ADONIS 002217599100312B 263 JIM06091 Determining the extent of labeling for tetramethylrhodamine protein conjugates David L. Meadows, Jules S. Shafer * and Jerome S. Schultz ** Department of Chemical Engineering, Uniuersity of Michigan, Ann Arbor, MI 48109, U.S.A. (Received 25 January 1991, revised received 21 June 1991, accepted 24 June 1991) A new, relatively simple, spectrophotometric technique has been developed which is useful for accurately determining the extent of chromophore labeling of proteins. Often the absorbance spectra and extinction coefficients of dye/protein conjugates are strongly affected by changes in the chromophore microenvironment that may occur at high dye/protein ratios. In the method being presented, the microenvironment effects have been significantly reduced by denaturing the dye/protein complex in 6 M guanidine hydrochloride prior to making the necessary spectrophotometric measurements. With this approach, extinction coefficients were obtained under native and denatured conditions for tetramethyl- rhodamine isothiocyanate (TRITC) when bound to a model protein receptor, the sugar binding protein concanavalin A (ConA). The extinction coefficients used for TRITC/ConA conjugates under native and denaturing conditions were 6.52 x 104 M-~ cm-~ and 6.96 x 104 M-1 cm-1, respectively. These values were obtained from a model dye complex formed between TRITC and E-amino-n-caproic acid which closely resembles the sidechain of lysine residues. Additional dye/ConA conjugates were prepared with tetramethylrhodamine succinimidyl ester (RHS) and eosin isothiocyanate (EITC), and the effects of microenvironment changes on these conjugates were examined. Extinction coefficients for these dyes in native and denaturing conditions, as a function of the degree of labeling, were not appreciably different indicating that changes in the microenvironment did not have a significant affect on the spectral properties of these two dyes. In summary, with this new approach it is quite easy to accurately determine the dye/protein ratio for TRITC conjugates. Also, it is expected that RHS would be a better dye than TRITC for protein conjugation because more accurate values for dye/protein ratios can be obtained under native conditions. Key words: Concanavalin A; Fluorescence; Labeling; Rhodamine; Eosin Correspondence to: D. Meadows, Allergan Pharmaceuticals, Inc., 2525 Dupont Drive, Irvine, CA 92715, U.S.A. * Present address: Department of Biochemistry, University of Michigan, Ann Arbor, MI, 48109 ** Present address: University of Pittsburgh, 911 William Pitt Union, Pittsburgh, PA 15260 Abbreciations: ACA, e-amino-n-caproic acid; ConA, concanavalin A; DMSO, dimethyl sulfoxide; EDTA, ethylenediaminete- traacetic acid; EITC, eosin isothiocyanate; GuHCI, guanidine hydrochloride; PBS, phosphate buffered saline i pConA, concanavalin A protomer; RHS, tetramethylrhodamine succinimidyl ester; Succ-ConA, succinylated concanavalin A; TRITC, tetramethylrho- damine isothiocyanate.