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The NH tautomerism of five Mg-free chlorophyll a and b derivatives 2-6 was studied utilizing NMR spectroscopy and molecular modeling. The results from the dynamic NMR measurements of the chlorins revealed that substituent effects contribute crucially to the free energy of activation (DeltaG(double dagger)) in the NH tautomeric processes. An intermediate tautomer for the total tautomeric NH exchange in a chlorin was observed for the first time, when the (1)H NMR spectra of chlorin e(6) TME (3) and rhodin g(7) TME (4) (TME = trimethyl ester) were measured at lower temperatures. The lower energy barriers (DeltaG(1)(double dagger)) obtained for the formation of the intermediate tautomers of 3 and 4, assigned to the N(22)-H, N(24)-H trans-tautomer, were 10.8 and 10.6 kcal/mol, respectively. The energy barrier (DeltaG(2)(double dagger) value) for the total tautomeric NH exchange in the five chlorins was found to vary from 13.6 kcal/mol to values higher than 18 kcal/mol. The lowest DeltaG(2)(double dagger) value (13.6 kcal/mol) was obtained for rhodochlorin XV dimethyl ester (2), which was the only chlorophyll derivative lacking the C(15) substituent. In the case of chlorins 4 and 5, the steric crowding around the methoxycarbonylmethyl group at C(15) raised the DeltaG(2)(double dagger) activation free-energy to 17.1 kcal/mol. However, the highest energy barrier with DeltaG(2)(double dagger) > 18 kcal/mol was observed for the NH exchange of pyropheophorbide a methyl ester (6), possessing the macrocycle rigidifying isocyclic ring E. Our results demonstrate that the steric strain, arising either from the steric crowding around the bulky substituent at C(15) or the macrocycle rigidifying isocyclic ring E, slows down the NH tautomeric process. We suggest that deformations in the chlorin skeleton are closely connected to the NH tautomeric exchange and that the exchange occurs by a stepwise proton-transfer mechanism via a hydrogen bridge.

Through partial bleaching of both visual pigment extracts and cell suspensions we show that the deep-sea stomiid Malacosteus niger, which produces far red bioluminescence, has two visual pigments within its retina which form a rhodopsin/porphyropsin pigment pair with lambda max values around 520 and 540 nm, but lacks the very longwave sensitive visual pigments (lambda max > 550 nm) observed in two other red light producing stomiids. The presence of only a single opsin gene in the M. niger genome was confirmed by molecular and cladistic analysis. To compensate for its apparently reduced longwave sensitivity compared to related species, the outer segments of M. niger contain additional pigments, which we identify as a mixture of defarnesylated and demetallated derivatives of bacteriochlorophylls c and d, that are used as a photosensitiser to enhance its sensitivity to longwave radiation.

Phospho- and glycolipids with mono- or polyunsaturated fatty acid chains, free and acylated steroids, carotenes and carotenoids, chlorophylls, and related pheophytins were detected in crude extracts by NMR spectroscopy without any need for prior separation. A broad range of molecules belonging to different lipid classes could be identified in one-dimensional 13C NMR spectra obtained by applying a polarization transfer pulse sequence (DEPT 135 degrees). Directly detected or 1H-detected two-dimensional heterocorrelated NMR experiments were performed to facilitate the assignment of peaks arising from carotenoids, unsaturated fatty acid chains, and chlorophylls. 13C NMR data of crude lipid extracts from the macroalagae Ulva rigida and Fucus virsoides are shown to yield an informative overview of their lipid content. NMR is thus proposed as a simple, nonselective, and nondestructive technique for the first screening of the main lipid classes in complex lipid mixtures.

An analytical separation method for tryptophan and its seven metabolites of the kynurenine pathway by high-voltage paper electrophoresis is presented. Anthranilic acid, 3-hydroxyanthranilic acid, kynurenic acid, kynurenine, nicotinic acid, quinolinic acid, xanthurenic acid and unmetabolized tryptophan are measured in urine. Using radioactive labelling and scintillation counting as a quantification method, the relative standard deviation varied from 3.5% to 14.4%, corresponding to kynurenine and nicotinic acid, respectively. The recovery of labelled tryptophan added to urine was 95%. An advantage of the electrophoretic method is the minor tailing of spots and, hence, a good resolution of the components. For the monovalent anions of the kynurenine pathway metabolites, a linear correlation (r = 0.9996) was found between the experimental relative electrophoretic mobility and the quantity M-2/3, where M is the molecular mass of the anion.

About 0.5% of the total cellular protein in the yeast Brettanomyces anomalus is membrane-bound cytochrome P-450, when this yeast is grown in the presence of 5% glucose as the main carbon and energy source. A partial purification of cytochrome P-450 by phase partition is described. Breakdown of yeast cell walls with microbial enzyme preparations led to extensive losses of this hemoprotein. Instead, by a carefully controlled mechanical breakage as much as 50% of the total cellular cytochrome P-450 could be recovered. During the solubilization of cytochrome P-450 from the cell homogenate with Triton X-100, the protective agents dithiothreitol, EDTA, and butylated hydroxytoluene prevented major losses of the hemoprotein. Applying a three-phase partition system (polyethylene glycol-Ficoll-dextran) to the solubilized whole cell homogenate in the presence of 1 M sodium chloride, followed by a precipitation of the top "oily layer" with 25% polyethylene glycol, a 25- to 60-fold enrichment of cytochrome P-450 was obtained. This corresponds to a specific content of 0.8-2.2 nmol of cytochrome P-450 per milligram of protein. Cytochrome b5 enriched (41%) to the PEG-Ficoll interphase, and NADPH-cytochrome c reductase and "cytochromes P-420" to the Ficoll and dextran phases. The polymer phase partition system thus serves as an excellent initial purification step of cytochrome P-450 without a need for the preparation of the microsomal fraction. Another advantage of the method is that it allows the simultaneous partial purification of cytochrome b5.

A method for the separation and identification of chlorophyll a and b, pheophytin a and b, pheophorbide a and b, chlorin e6, rhodin g7, and the corresponding chlorophyll a' and b' derivatives, as well as the saponification products of pheophorbide a and b, by thin-layer chromatography on commercial cellulose layers on the micro-scale has been developed. Two solvent systems were used: light petroleum (b.p.60-80 degrees)-pyridine (9:1, v/v) and n-heptane-pyridine (7:3, v/v). The former was suitable for chlorophylls, pheophytins and pheophorbides, and the latter for pheophorbides, chlorin, rhodin and their esters. The separation of the derivatives was good and no chemical alteration of the derivatives could be observed. The method is rapid and easy to use and is therefore suitable for checking the purity of derivatives during the preparation of chlorophylls and their derivatives.