A spectrophotometric method for determination of fluorophore-to-protein ratios in conjugates of the blue fluorophore 7-amino-4-methylcoumarin-3-acetic acid (AMCA)

7-Amino-4-methylcoumarin-3-acetic acid (AMCA) has been found to be a useful fluorophore for immunofluorescence. The present study describes a spectrophotometric method for determining the ratio of moles AMCA to moles protein (or the f/p ratio) in an AMCA-conjugated IgG. The concentration of a substance absorbing light can be determined spectrophotometrically using Beer's Law: Absorbance = Concentration x Extinction coefficient. From Beer's law, one can derive the following formula for determining the f/p ratio of AMCA-IgG conjugates: f/p = ((ε₃₅₀(AMCA)) · A₂₈₀ - (ε₂₈₀(AMCA)) · A₃₅₀) ((̇₂₈₀(IgG)) · A₃₅₀ - (̇₃₅₀(IgG)) · A₂₈₀) where A is the optical density of the conjugate at the given wavelength and ε is the extinction coefficient of a substance at the wavelength specified. Using conjugates of model proteins, it was found that the extinction coefficients of the AMCA moiety of AMCA-conjugated protein were 1.90 x 10⁴ at 350 nm and 8.29 x 10³ at 280 nm. Similarly, it was found that the extinction coefficients of swine IgG were 1.56 x 10³ at 350 nm and 1.26 x 10⁵ at 280 nm. Thus, for AMCA-conjugated swine IgG: f/p = ((1.90 x 10⁴) · A₂₈₀ - (8.29 x 10³) · A₃₅₀) ((1.26 x 10⁵) · A₃₅₀ - (1.56 x 10³) · A₂₈₀). Based on this formula, the f/p ratios of some AMCA-IgG conjugates useful for immunohistochemistry have been found to range between 6 and 24.