High-performance liquid chromatography of non-polar retinoid isomers

Nöll, Gottfried N.; Becker, Christian

doi:10.1016/s0021-9673(00)00232-6

Cited by 9 publications

(5 citation statements)

References 10 publications

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“…A total of 500 μl of hexane was then added, and samples were agitated for 2 min and subsequently centrifuged for 1 min. The resulting organic layer was transferred to a sample vial with a Pasteur pipet and immediately analyzed by HPLC (Waters 600E multisolvent delivery system with a Water 2487 dual wavelength detector) in a method adapted from recent work by G. Noll and C. Becker . Two Prep Nova-Pak HR Silica columns (3.9 × 300 mm 2 , Waters, Milford, MA) in series provided the stationary phase.…”

Section: Methodsmentioning

confidence: 99%

“…The resulting organic layer was transferred to a sample vial with a Pasteur pipet and immediately analyzed by HPLC (Waters 600E multisolvent delivery system with a Water 2487 dual wavelength detector) in a method adapted from recent work by G. Noll and C. Becker. 19 Two Prep Nova-Pak HR Silica columns (3.9 × 300 mm 2 , Waters, Milford, MA) in series provided the stationary phase. The mobile phase, flowing at 1.5 mL/min, was composed of 93% hexane and 7% tert-methyl butyl ether (TBME).…”

Section: Methodsmentioning

confidence: 99%

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Characterization of the Branched-Photocycle Intermediates P and Q of Bacteriorhodopsin

et al. 2002

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The bacteriorhodopsin branched-photocycle intermediates P and Q are studied with respect to chromophore isomeric content, photochemical origin, kinetic heterogeneity, and photoreversibility. These blue-shifted species are compared to products with similar spectroscopic properties generated via thermal denaturation. We observe that the thermal and photochemical species differ in both isomeric content and binding site environment. Sequential two-photon activation of glycerol suspensions of bacteriorhodopsin containing low concentrations of water (<15% v/v water/glycerol) form high yields of P state but almost no Q state. This observation is attributed to the role that water plays in the hydrolysis reaction that converts the P state to Q. Relatively large photostationary state populations of both intermediates can be generated in both 85% v/v glycerol suspensions and polyacrylamide gels. At ambient temperature, both intermediates can be fully converted back to bR with blue light. Chromophore extraction and HPLC analysis reveal that P, Q, and the spectrally similar thermal products have a 9-cis retinal chromophore. The thermal products are also found to contain small amounts of the 7-cis isomer. Time-resolved spectroscopy reveals that the P state is actually comprised of two components with maximum absorptivities at 445 and 525 nm. The molar absorptivities of the chromophore band maxima of the P and Q states in an 85% v/v glycerol/water suspension at pH 7 are ε(P 445) = 47 000, ε(P 525) = 39 000, and ε(Q) = 33 000 M-1 cm-1. Kinetic analyses support previous studies indicating that the P state is formed predominantly from the O state, which is consistent with the branched-photocycle model. However, other paths to the P state are possible, including direct excitation of the small population of blue membrane present in solution. We examine the hypothesis that the branched-photocycle evolved as a photochromic sunscreen for UVA and UVB protection.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Characterization of the Branched-Photocycle Intermediates P and Q of Bacteriorhodopsin

et al. 2002

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“…HPLC was carried out on a Jasco PU-980 equipped with a Jasco UV-975 UV detector. Samples were separated on a silica column (5 μm, 250 mm × 4.6 mm, Agilent Technology) with 98:2 (v/v) hexane/ tert -butyl methyl ether as the mobile phase for isocratic elution . Peak fractions were collected, and the solvent was evaporated under a nitrogen stream.…”

Section: Methodsmentioning

confidence: 99%

“…Samples were separated on a silica column (5 μm, 250 mm × 4.6 mm, Agilent Technology) with 98:2 (v/v) hexane/tert-butyl methyl ether as the mobile phase for isocratic elution. 23 Peak fractions were collected, and the solvent was evaporated under a nitrogen stream. For UV/vis spectroscopy, the residual oil film of dried peak fractions was dissolved in ethanol and analyzed with an Evolution Array UV/vis spectrophotometer (Thermo Scientific).…”

Section: ■ Experimental Sectionmentioning

confidence: 99%

Human Cellular Retinaldehyde-Binding Protein Has Secondary Thermal 9-cis-Retinal Isomerase Activity

Bolze¹,

Helbling²,

Owen

et al. 2013

J. Am. Chem. Soc.

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Cellular retinaldehyde-binding protein (CRALBP) chaperones 11-cis-retinal to convert opsin receptor molecules into photosensitive retinoid pigments of the eye. We report a thermal secondary isomerase activity of CRALBP when bound to 9-cis-retinal. UV/VIS and 1H-NMR spectroscopy were used to characterize the product as 9,13-dicis-retinal. The X-ray structure of the CRALBP mutant R234W:9-cis-retinal complex at 1.9 Å resolution revealed a niche in the binding-pocket for 9-cis-aldehyde different from that reported for 11-cis-retinal. Combined computational, kinetic, and structural data lead us to propose an isomerization mechanism catalyzed by a network of buried waters. Our findings highlight a specific role of water molecules in both CRALBP-assisted specificity towards 9-cis-retinal and its thermal isomerase activity yielding 9,13-dicis-retinal. Kinetic data from two point mutants of CRALBP support an essential role of Glu202 as the initial proton donor in this isomerization reaction.

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“…NPC was also employed successfully for the resolution of (a) four configurational isomers of a steroidal calyx pyrrole [45], (b) regio-and stereoisomers of eicosanoids [46], (c) retinal and retinol isomers [47], and (d) several E/Z isomers pairs of vitamin A [48].…”

Section: Separation Of Isomersmentioning

confidence: 99%