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To investigate the structural modulation of ligands and their interaction in the active-site nanospace when they form charge-transfer (CT) complexes with D-amino acid oxidase (DAO) in three redox states, we compared Ramanbands of the ligands in complex with DAO with those of ligands free in solution. Isotope-labeled ligands were synthesized for assignments of observed bands. The COO- stretching of ligands observed around, 1,370 cm - 1 downshifted by about 17 cm - 1 upon complexation with oxidized, semiquinoid and reduced DAO, except for the case of reduced DAO-N-methylisonicotinate complex (8 cm - 1 downward shift); the interaction mode of the carboxylate group with the guanidino group of Arg283 and the hydroxy moiety of Tyr228 of DAO is similar in the three redox states. The C=N stretching mode (1,704 cm - 1 ) of Δ 1 -piperideine-2-carboxy-late (D1PC) downshifted to 1,675 and 1,681 cm - 1 upon complexation with reduced and semiquinoid DAO, respectively. The downward shifts indicate that the C=N bond is weakened upon the complexation. This is probably due mainly to charge-transfer (CT) interaction between D1PC and semiquinoid or reduced flavin, i.e., the partial electron donation from the highest occupied molecular orbital (HOMO) of reduced flavin or a singly occupied molecular orbital (SOMO) of semiquinoid flavin to the lowest unoccupied molecular orbital (LUMO), an antibonding orbital, of D1PC. This speculation was supported by the finding that the magnitude of the shift is smaller by 5 cm - 1 (observed at 1,680 cm - 1 ) in the case of reduced DAO reconstituted with 7,8-Cl 2 -FAD, whose reduced form has lower electron-donating ability than natural reduced FAD. The amount of electron flow was estimated by applying the theory of Friedrich and Person [(1966) J. Chem. Phys. 44, 2166-2170] to these complexes; the amounts of charge transfer from reduced FAD and reduced 7,8-Cl 2 -FAD to D1PC were estimated to be about 10 and 8% of one electron, respectively, in the CT complexes of reduced DAO with D1PC.

We characterized differences in biochemical composition of the vitreous of different animal species with respect to small molecule constituents. Vitreous samples were extracted from sheep, pig, Dutch Belted rabbits, and New Zealand white rabbits. The vitreous samples were investigated for acetylcholinesterase (AChE) activity and, in addition, were subjected to metabolomics determination using mass spectrometry. AChE activity varied across the species investigated with greater activity noted in larger animals. Principal component analysis demonstrated species differentiation in relation to metabolomic profile. Key peaks identified the importance of animal diet on small molecule composition of the vitreous. Our results highlighted principal and consistent differences in small molecule composition and enzymatic activity of the vitreous depending on species. Interesting differences were demonstrated, showing that diet potentially can impact on components of and metabolites contained within the vitreous. Material will be exchanged between vascular and retinal tissue with the vitreous compartment and as a nonvascular, slowly equilibrating “sink” might reflect changes in transporter activity. As a first step, understanding the differences in the metabolic profile of vitreous from different species may impact interpretation of such activity across different species.