Interaction of Hypericin with Serum Albumins: Surface-enhanced Raman Spectroscopy, Resonance Raman Spectroscopy and Molecular Modeling Study¶

Abstract

Surface-enhanced Raman spectroscopy, resonance Raman spectroscopy and molecular modeling were employed to study the interaction of hypericin (Hyp) with human (HSA), rat (RSA) and bovine (BSA) serum albumins. The identification of the binding site of Hyp in serum albumins as well as the structural model for Hyp/ HSA complex are presented. The interactions mainly reflect: (1) a change of the strength of H bonding at the N1-H site of Trp; (2) a change of the Trp side-chain conformation; (3) a change of the hydrophobicity of the Trp environment; and (4) a formation of an H-bond between the carbonyl group of Hyp and a proton donor in HSA and RSA which leads to a protonated-like carbonyl in Hyp.Our results indicate that Hyp is rigidly bound in IIA subdomain of HSA close to Trp214 (distance 5.12 Å between the centers of masses). In the model presented the carbonyl group of Hyp is hydrogen bonded to Asn458. Two other candidates for hydrogen bonds have been identified between the bay-region hydroxyl group of Hyp and the carbonyl group of the Trp214 peptidic link and between the peri-region hydroxyl group of Hyp and the Asn458 carbonyl group. It is shown that the structures of the Hyp/HSA and Hyp/RSA complexes are similar to, and in some aspects different from, those found for the Hyp/BSA complex. The role of aminoacid sequence in the IIA subdomains of HSA, RSA and BSA is discussed to explain the observed differences. ¶Posted on the website on

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“…The concentration ratio of 4:1 was found to be optimal (Figure 4), as any further increase in hypericin concentration led to fluorescence self-quenching. This methodology of conjugating hypericin with dBSA to prevent fluorescence quenching by nanostars was corroborated by another group by [26] who investigated the conjugation site of hypericin within serum albumins and reported that hypericin binds with the deeply localized IIA sub-domain of BSA. This study also investigated SERS of hypericin when conjugated with BSA-hypericin complex.…”

“…This study also investigated SERS of hypericin when conjugated with BSA-hypericin complex. A strong reduction in intensity of hypericin's Raman bands when in complex with BSA was observed using SERS spectroscopy which is indicative of its greater distance from nanoparticle's surface resulting from its conjugation with BSA [26]. Thus, conjugating hypericin with BSA provided the dual purpose of stabilizing nanostars and also localizing hypericin in deep pockets within BSA, enabling hypericin to retain its fluorescence.…”

“…Unreacted sodium borohydride was removed by spontaneous decomposition [25]. Hypericin was added to denatured BSA (dBSA) and left overnight for incubation [26]. The dBSA:Hyp complex was purified by removing unbound hypericin using Nanosep™ 10000 MWCO OMEGA membrane (Pall Life Sciences).…”