Transient Absorption Spectroscopy for Determining Multiple Site Occupancy in Drug−Protein Conjugates. A Comparison between Human and Bovine Serum Albumins Using Flurbiprofen Methyl Ester as a Probe

Abstract

Laser flash photolysis (LFP) has been used to determine the degree of binding of (S)- or (R)-flurbiprofen methyl ester (FBPMe) to human and bovine serum albumins (HSA and BSA, respectively). Regression analysis of the triplet decay of the drug (lambda = 360 nm) in the presence of the proteins led to a satisfactory fitting when considering a set of three lifetimes; the corresponding Afree, AI and AII preexponential coefficients can be correlated with the presence of FBPMe in the bulk solution and within the two known binding sites. The most remarkable differences between HSA and BSA were found under nonsaturating conditions; thus, when the [FBPMe]/[SA] ratio was 1:1, all the drug was bound to HSA, whereas 20-30% of it remained free in the bulk solution in the presence of BSA. The LFP approach was also applicable to the study of more complex FBPMe/HSA/BSA mixtures; the obtained results were in good agreement with the previous findings in FBPMe/HSA and FBPMe/BSA systems. This suggests the possibility of making use of the transient triplet-triplet absorption for investigating the distribution of a drug between several compartments in different host biomolecules.