In situ transient spectroscopy for the study of glucuronidase activity within serum albumin.

Laser flash photolysis (LFP) has shown to be an efficient technique for in situ determination of the glucuronidase activity of human serum albumin (HSA). After incubation of the steroisomeric flurbiprofen glucuronides (FBPGluc) during regular time intervals at the selected temperatures, in the presence of protein, regression analysis was applied to the triplet decay at lambda=360 nm. This led to a satisfactory fitting when considering a set of four lifetimes; the corresponding preexponential coefficients AIFBP, AIIFBP, AFFBPGluc, and ABFBPGluc can be correlated with the presence of flurbiprofen (FBP) within the two known binding sites (I and II), together with FBPGluc free in solution (F) and bound (B) to the protein. The new methodology based on LFP of glucuronides in the presence of HSA is fast, experimentally straightforward, and does not involve any workup. This suggests the possibility of making use of the transient triplet-triplet absorption for investigating the enzymatic-like activity of different host biomolecules and at the same time determining the distribution of the generated drug between several compartments in the protein.

Use of triplet excited States for the study of drug binding to human and bovine serum albumins.

The triplet excited states of (S)- and (R)-flurbiprofen (FBP) have been used as reporters for the microenvironments experienced within the binding sites of human and bovine serum albumins. Regression analysis of triplet decay provides valuable information on the degree of protection that these excited states are afforded from attack by a second FBP molecule, oxygen, or other reagents. The multiexponential fitting of these decays can be satisfactorily correlated with the distribution of the drug among the two binding sites and its presence as the noncomplexed form in the bulk solution. This assignment has been confirmed by using (S)-ibuprofen or capric acid as selective site II replacement probes. Triplet lifetimes and site occupancy are sensitive to the type of serum albumin employed (human versus bovine). Finally, the binding behaviour of (S)- and (R)-FBP exhibits little stereoselectivity.