Determination of the molar absorbance coefficient of bound tetramethyl rhodamine isothiocyanate relative to fluorescein isothiocyanate

Abstract

Listen

Translate article

Since the introduction of tetramethyl rhodamine isothiocyanate (TRITC) as a fluorochrome in immunofluorescence by Hiramoto et al. (1958), it has been used with success either alone or in combination with fluorescein isothiocyanate (FITC) (Cebra and Goldstein, 1965,; Nairn et al., 1969; Hijmans et al., 1971 ; Loor et al., 1972). For preparation and standardization of TRITC conjugates, measurement of the number of fluorochrome molecules per molecule of protein (F/P ratio) is obligatory. For FITC this ratio has been shown to be important in determining the behaviour of a given conjugate in immunofluorescence (Hebert et al., 1967, and Beutner, 1971). The F/P ratio can be determined only if the molar absorbance coefficient (e) of the fluorophore in bound form is known. McKinney et al. (1966) measured the e of bound FITC by reacting a given amount of FITC with excess bovine serum albumin (BSA) which resulted in over 95% binding of the FITC to BSA. The absorbance of a diluted aliquot of this bound FITC was compared with that of the original FITC solution. Tengerdy and Chang (1966) measured the uptake of FITC by conalbumin using FITC absorbed to Celite. Molar absorbance coefficients were obtained for FITC in solution and in bound form relative to a disodium fluorescein standard. These methods appear not to be suitable for TRITC because the purity of the reagent is unknown and no accepted standard for TRITC exists. Furthermore, during the reaction of TRITC with protein, precipitation of the dye often takes place. In this communication, we present a method by which eboun d TRITC is determined relative to eboun d FITC.