Determining the extent of labeling for tetramethylrhodamine protein conjugates

Abstract

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 tetramethylrhodamine 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 × 10 4 M −1 cm −1 and 6.96 × 10 4 M −1 cm −1, respectively. These values were obtained from a model dye complex formed between TRITC and ϵ-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.