Determining molar absorption coefficients for chromoproteins using relative actinometry with transient absorption...

Determining molar absorption coefficient for chromoprotein samples containing a significant fraction of apoprotein is problematic since conventional protein quantification methods, such as the Bradford assay, determine the total amount of protein. This leads to underestimation of molar absorption coefficient. Using Orange Carotenoid Protein (OCP) as a model system, we demonstrate an alternative approach employing the relative actinometry method and femtosecond transient absorption spectroscopy. By comparing the ground state bleaching bands of OCP and β-carotene recorded immediately after the excitation, before the onset of decay, we determined a molar absorption coefficient that is representative of the holoprotein. β-carotene serves as a well-characterized reference compound with photophysics similar to that of OCP, enabling error cancellation. We obtained a value of 110,000 M^- 1 cm^-1 at the absorption maximum, significantly higher than previously reported values (63,000 M^-1 cm^-1 and 87,270 M^-1 cm^- 1). The method can also be applied to other proteins where conventional approaches fail due to a significant amount of apoprotein.