Determination of LMF Binding Site on a HSA-PPIX Complex in the Presence of Human Holo Transferrin from the Viewpoint of Drug Loading on Proteins.

Sattar, Zohreh; Saberi, Mohammad Reza; Chamani, Jamshidkhan

Holo transferrin (TF) and the natural complex of human serum albumin and protoporphyrin IX (HSA-PPIX) are two serum carrier proteins that can interact with each other. Such an interaction may alter their binding sites. In this study, fluorescence spectroscopy, as well as zeta potential and molecular modeling techniques, have been used to compare the complexes (HSA-PPIX)-LMF and [(HSA-PPIX)-TF]-LMF. The Ka1, Ka2, values of (HSA-PPIX)-LMF and [(HSA-PPIX)-TF]-LMF were 1.1×105 M−1, 9.7×106 M−1, and 2.0×104 M−1, 1.8×105 M−1, respectively, and the n1, n2 values were respectively 1.19, 1.53 and 1.17, 1.65. The second derivative of the Trp emission scan of (HSA-PPIX)-LMF exhibited one negative band at 310 nm, whereas for the [(HSA-PPIX)-TF]-LMF system, we observed one negative band at 316 nm indicating an increase in polarity around Trp. The effect of TF on the conformation of (HSA-PPIX)-TF was analyzed using three-dimensional fluorescence spectroscopy. The phase diagram indicated that the presence of a second binding site on HSA and TF was due to the existence of intermediate structures. Zeta potential analysis showed that the presence of TF increased the positive charges of the HSA-PPIX system. Site marker experiments revealed that the binding site of LMF to HSA-PPIX changed from Sudlow's site IIA to Sudlow's site IIIB in the presence of TF. Moreover, molecular modeling studies suggested the sub-domain IIIB in HSA as the candidate place for the formation of the binding site of LMF on the (HSA-PPIX)-TF complex.

TRANSFERRIN; BINDING sites; SERUM albumin; PROTOPORPHYRINS; FLUORESCENCE spectroscopy; ZETA potential; DRUG delivery systems; MOLECULAR models

PLoS ONE, 2014, Vol 9, Issue 1, p1

1932-6203

Academic Journal

10.1371/journal.pone.0084045