Robert Boykins CBER - Food and Drug Administration Office: 301-496-2902 FAX: E-mail: robert.boykins@fda.hhs.gov Job Title: Staff Scientist B.Sc. in Chemistry from the North Carolina A&T State University

Speaker: Robert Boykins, Center for Biologics Evaluation and Research (CBER), Food and Drug Administration, 8800 Rockville Pike, Bethesda, MD 20892

Topic: O-Raffinose Crosslinked Hemoglobin Lacks Site-Specific Chemistry in the Central Cavity: Structural and Functional Consequences of beta chain Cysteine(93) Modification

Place: Building 549, Auditorium, NCI at Frederick, Frederick, MD

Time: Tuesday, February 21, 2006, at 2:00 PM

Abstract: Reacting human deoxyHbA0 with oxidized raffinose (O-raffinose), a trisaccharide, results in a low oxygen affinity "blood substitute," stabilized in a noncooperative T-conformation and possesses readily oxidizable rhombic heme. In this study, we fractionated the O-raffinose-modified HbA0 heterogeneous polymer (O-R-PolyHbA0) into six distinct fractions with a molecular weight distribution ranging from 64 to approximately 600 kDa using size-exclusion chromatography (SEC). Oxygen equilibrium and kinetics binding parameters of all fractions were nearly identical, reflecting a lack of heterogeneity in ligand binding properties among O-R-PolyHbA0 species (Hill coefficient n equal to 1.0). Several mass spectrometry techniques were used to evaluate undigested and digested HbA0, O-R-PolyHbA0, and O-R-PolyHbA0 fractions. Proposed sites of intramolecular crosslinking (i.e., beta1Lys82, beta2Lys82, and beta1Val1) were not found to be the predominant site of crosslinking within the central cavity. Intermolecular crosslinking with O-raffinose results in no discernible site of amino acids modifications with the exception of beta93Cys and alpha104Cys. Based on accessible surface area (ASA) calculations in intact deoxyHbA0, slight conformational changes are required to allow for the S on alpha104Cys to be modified during the reaction with O-raffinose or its partially oxidized product(s). The stabilization of HbA0 in the T-conformation may not be a direct correlate of O-raffinose induced changes, but an indirect consequence of changing hydration in the water-filled central cavity and/or the distal heme pocket leading in the latter case to accelerated iron oxidation. Structural data presented here when taken together with the oxidative instability of O-R-PolyHbA0 may provide some basis for the reported toxicity of this oxygen carrier.

The slides to this seminar are in a 1.4 Megabyte PDF file, which can be opened and read by using the free Adobe Acrobat Reader®.

This work has been published: R. A. Boykins, P. W. Buehler, Y. Jia, R. Venable, and A. I. Alayash, Proteins: Structure, Function, and Bioinformatics 59, 840-855 (2005).