Novel combinations of experimental and computational analysis tested on the binding of metalloprotoporphyrins to albumin.

The evidence that Human Serum Albumin (HSA) binds metal ions and organometallic compounds has generated interest in its physiological role as a metalloprotein and as a vehicle for synthetic biology applications (e.g., synthetic blood and solar energy conversion). HSA has been shown to bind metallo-porphyrins, however, the structural details of such interactions are available only for the HSA:heme complex. A typical challenge for studying the interaction of proteins with metalloporphyrins is the poor solubility of the ligands that affect the characterization the complexes. The manuscript shows that a combination of dialysis and centrifugation yields aqueous solutions that contain >90% HSA:porphyrin complexes and virtually eliminate aggregated ligands. The removal of aggregates increases the quality of the optical spectroscopy data which, in turn, yield more accurate binding constants (~0.1 and 2.1 × 10(6) M(-1)) and reveal FRET between Trp214 and the porphyrins. The Trp-porphyrin distance was estimated to be within the 28-34 à range and was used to guide the search of binding sites through a novel feedback approach with docking simulations. Results suggest while some protoporphyrins (metal-free, Fe(III)PPIX and Mg(II)PPIX) bind HSA at the heme site, others (Zn(II)PPIX, Mn(III)PPIX and Sn(IV)PPIX) are more likely to bind the Cys34.