Inaccuracy of the Bradford method for the determination of protein concentration in steroid-horseradish peroxidase conjugates

Conjugates between horseradish peroxidase and steroid molecules were prepared with different steroids (progesterone, testosterone, cortisol, estradiol) and different reaction mole ratios. These conjugates were characterised by liquid chromatographic analysis to check purity grade, stability and the presence of polymeric compounds, and by spectrophotometric measurements to obtain values of the average substitution ratio and of protein concentration by the absorbance corresponding to the Soret band at 402 nm. The results obtained showed that the Bradford method, based on interactions between Coomassie Brilliant Blue G-250 and the amine groups on the protein surface, lead generally to an underestimation of protein concentration in steroid-horseradish peroxidase conjugates.