The main pigment of the dormant Mycobacterium smegmatis is porphyrin.

Upon transition of Mycobacterium smegmatis into the dormant state, accumulation of a dark brown fluorescent pigment was observed. This pigment gave bright red fluorescence in both cells and the culture medium. Based on 1H-NMR, MALDI and UV spectra, the fluorescent compounds, extracted from the culture medium as well as from the dormant cells, were concluded to be a mixture of free coproporphyrin III and uroporphyrin III and their corresponding methyl esters. A possible significance of porphyrin pigment accumulation in the dormant cells is discussed.

Direct assay of uroporphyrin and coproporphyrin in human urine by reverse-mode field amplified sample injection-sweeping and micellar electrokinetic chromatography.

In this study, an on-line stacking capillary electrophoresis (CE) method, reverse-mode field amplified sample injection equipped with sweeping micellar electrokinetic chromatography (RMFASI-sweeping MEKC), was established for direct determination of uroporphyrin and coproporphyrin in human urine. Porphyrins playing a very important role in the biosynthesis of heme, chlorophyll and other important enzymes are a series of important molecules in organism. Therefore, determination of porphyrin metabolites, uroporphyrin and coproporphyrin, was very important for clinical survey of some diseases. In this study, the urine sample after simple dilution could be directly analyzed by this on-line stacking CE method. The optimal CE separation buffer was 70 mM phosphate buffer at pH 3. Before sample injection, a water plug was introduced (2.5 psi for 10s), and then the samples were loaded by electrokinetic injection (-10 kV, 200 s). Finally, the phosphate buffer (70 mM, pH 3) containing 100mM SDS was served as the sweeping buffer to stack and separate the analytes at -20 kV. The calibration curves were linear over a range of 15-200 ng/ml for uroporphyrin, and 300-1000 ng/ml for coproporphyrin. The coefficient of correlation (r) in intra-batch (n=5) and inter-batch (n=5) analysis was above 0.983. The LODs (S/N=3) were 5 ng/ml for uroporphyrin, and 100ng/ml for coproporphyrin. The absolute values of relative standard deviation (RSD) and relative error (RE) in intra-batch (n=5) and inter-batch (n=5) assays were less than 8.6% showing the good precision and accuracy. The stacking method was successfully applied in real urine sample and feasible for serving as a tool for detection of uroporphyrin and coproporphyrin in clinical.

An enzyme-trap approach allows isolation of intermediates in cobalamin biosynthesis.

The biosynthesis of many vitamins and coenzymes has often proven difficult to elucidate owing to a combination of low abundance and kinetic lability of the pathway intermediates. Through a serial reconstruction of the cobalamin (vitamin B(12)) pathway in Escherichia coli and by His tagging the terminal enzyme in the reaction sequence, we have observed that many unstable intermediates can be isolated as tightly bound enzyme-product complexes. Together, these approaches have been used to extract intermediates between precorrin-4 and hydrogenobyrinic acid in their free acid form and permitted the delineation of the overall reaction catalyzed by CobL, including the formal elucidation of precorrin-7 as a metabolite. Furthermore, a substrate-carrier protein, CobE, that can also be used to stabilize some of the transient metabolic intermediates and enhance their onward transformation, has been identified. The tight association of pathway intermediates with enzymes provides evidence for a form of metabolite channeling.

Rapid analysis of porphyrins at low ng/l and microg/l levels in human urine by a gradient liquid chromatography method using octadecylsilica monolithic columns.

Rapid gradient RP-HPLC method with fluorimetric detection for trace analysis of diagnostically significant porphyrins in human urine was developed for clinical and diagnostic purposes. Results show that optimized high-pressure gradient elution and monolithic column Chromolith SpeedRod RP18e enabled separation of seven urine porphyrins including baseline separation of I and III positional isomers of uro- and coproporphyrins within 3.2 min. Problems associated with high metal cation complexing ability of the analytes and common stainless steel based instrumentation were substantially reduced by use of 0.1 mol/l ammonium citrate buffer (pH 5.47) and methanol as a mobile phase components. Good reproducibilities of retention times (within +/- 0.36% RSD) and peak areas (from +/- 0.6 to +/- 2.5% RSD) at 5-20 microg/l level of the analytes were achieved. Determined LOQ (10 x S/N) values of diagnostically important porphyrins using fluorimetric detection (ex.405 nm/em.620 nm) were 82 pmol/l (65 ng/l, 1.30 pg/injection) for uroporphyrin I, 44 pmol/l (33 ng/l, 0.66 pg/injection) for uroporphyrin III, 50 pmol/l (40 ng/l, 0.80 pg/injection) for coproporphyrin I and 47 pmol/l (39 ng/l, 0.78 pg/injection) for coproporphyrin III. Attained LOQ concentration level is approximately 20-120 times lower than concentration of porphyrins in a urine of healthy person. Calculated LOD's (3 x S/N) were at a low ng/l levels, what enabled quantification of carry-over effect to be from 2.0% to 0.2% in each of three consecutive blank runs and from 2.5% to 7% in total after injection of mixed standard of porphyrins with 5-20 microg/l concentrations. Recovery of porphyrins at low microg/l concentration levels was from 93% to 97.5%. Devised method increases productivity of clinical laboratory from 2 to 10 times in dependence of duration of currently used method.

Arsenite alters heme synthesis in long-term cultures of adult rat hepatocytes.

Arsenite (As[III]) effects on the intermediate steps of heme biosynthesis were studied in adult rat hepatocytes seeded on a feeder layer of 3T3 cells (3T3-hepatocytes) and maintained for 2 weeks with culture medium non-supplemented or supplemented with 150 microM 5-aminolevulinic acid (ALA). The activities of the intracellular enzymes porphobilinogen deaminase (PBG-D), uroporphyrinogen III synthase (UROIII-S), and uroporphyrinogen III decarboxylase (URO-D), and the intermediary uroporphyrins (URO), coproporphyrins (COPRO) and protoporphyrin IX (PROTO) were determined in these cultures. The 3T3-hepatocytes maintained the activities of PBG-D, UROIII-S and URO-D during 2 weeks and ALA addition to the culture medium increased PBG-D (2-3-fold) and UROIII-S (50%) activities and porphyrin production, which accumulated as PROTO. Exposure to 3.9 microM As(III) inhibited UROIII-S activity (down to 34%), and PBG-D and URO-D activities to a lower extent; these effects were magnified by ALA supplementation. As(III) also produced an intracellular accumulation and a decreased excretion of PROTO, and a 31% reduction of the COPRO/URO ratio in the culture medium. Additionally, As(III) caused cytoplasmic vacuolization and lipid accumulation. Our results show that As(III) exposure selectively inhibits several intermediary enzymes of heme metabolism and affects the intra- and extracellular content of porphyrins and their ratio in the culture medium. They also confirm that 3T3-hepatocytes are a suitable in vitro model to study hepatic heme metabolism and its alterations by hepatotoxic chemicals.

Preparation and separation of hydroxy derivatives of uroporphyrinogen I by high-performance liquid chromatography with electrochemical detection.

The preparation and high-performance liquid chromatography (HPLC) separation of meso-hydroxyuroporphyrinogen I, hydroxyacetic acid uroporphyrinogen I and beta-hydroxypropionic acid uroporphyrinogen I is described. meso-Hydroxyuroporphyrin I, hydroxyacetic acid uroporphyrin I and beta-hydroxypropionic acid uroporphyrin I were isolated from the urine of a patient with congenital erythropoietic porphyria. The porphyrins were reduced to the corresponding porphyrinogens with 3% (w/w) Na/Hg amalgam. The hydroxy porphyrinogens were separated on a Hypersil ODS column with 4% (v/v) acetonitrile in 1 M ammonium acetate buffer, pH 5.16, containing EDTA (0.27 mM) as the mobile phase, and detected electrochemically. Reduction of meso-hydroxyuroporphyrin I and hydroxyacetic acid uroporphyrin I, followed by HPLC analysis, showed that, in addition to the expected formation of meso-hydroxyuroporphyrinogen I and hydroxyacetic uroporphyrinogen I, respectively, uroporphyrinogen I was also produced. Reduction of beta-hydroxypropionic acid uroporphyrin I, however, gave beta-hydroxypropionic acid uroporphyrinogen I, acrylic acid uroporphyrinogen I and uroporphyrinogen I as the products. The peaks were identified by conversion into the porphyrin methyl esters and analysed by liquid secondary-ion mass spectrometry.

New intermediates in the B12 pathway.

Vitamin B12 has a complex structure which represents one of the most challenging biosynthetic problems in Nature. Exciting progress has been made by combining the techniques, approaches and strengths of chemistry, spectroscopy and biology. Most of the advances until recently came from experiments based either on labelling simpler precursors with radioactive isotopes followed by controlled degradation of the labelled products, or on the use of stable isotopes, 13C in particular, because it can be detected and its environment can be studied by NMR spectroscopy. These experiments imposed heavy demands on synthesis which provided the specifically labelled starting materials. More recently, the powerful methods of genetics and molecular biology have been added to the armoury, leading to another massive surge forward by allowing the preparation, through gene overexpression, of large quantities of the enzymes of the biosynthetic pathway. Equally important has been the generation of mutant forms of B12-producing organisms in which the biosynthetic pathway is blocked at specific points. Here I focus on the latest advances. The structures of the newly discovered intermediates are described and some of the chemistry involved is explored. In conclusion, the presently known pathway to vitamin B12 is reviewed.

The final step in the biosynthesis of hydrogenobyrinic acid is catalyzed by the cobH gene product with precorrin-8x as the substrate.

The final enzymatic reaction in the conversion of precorrin-6x to hydrogenobyrinic acid by cell-free protein preparations from Pseudomonas denitrificans was shown to be inhibited by hydrogenobyrinic acid. Use was made of this property to prepare the last biosynthetic precursor of hydrogenobyrinic acid, named precorrin-8x. Double-labeling experiments, mass spectrometry, and UV-visible light spectroscopy studies established that precorrin-8x was at the oxidation level of a corrin and differed from precorrin-6x by two additional methyl groups (presumably at C-5 and C-15) and decarboxylation of the acetic acid side chain at C-12. Precorrin-8x was not a corrin but had the same mass as hydrogenobyrinic acid, thus showing that this latter compound is synthesized from the former by a rearrangement. The enzyme catalyzing this rearrangement was purified 80-fold to homogeneity from a recombinant strain of P. denitrificans, sequenced at its N terminus, and shown to be encoded by the cobH gene. It was identical to the previously described hydrogenobyrinic acid-binding protein (F. Blanche, D. Thibaut, D. Frechet, M. Vuilhorgne, J. Crouzet, B. Cameron, G. Müller, K. Hlineny, U. Traub-Eberhard, and M. Zboron, Angew. Chem. Int. Ed. Engl. 29:884-886, 1990). This enzyme had a Km of 0.91 +/- 0.04 microM and a Vmax of 230 nmol h-1 mg-1 at pH 7.7 and was competitively inhibited by hydrogenobyrinic acid with a Ki of 0.17 +/- 0.01 microM. It is proposed that the cobH gene product is a mutase which transfers the methyl group from C-11 to C-12.

Identification of peroxyacetic acid uroporphyrin I in the urine of patients with congenital erythropoietic porphyria by liquid chromatography and mass spectrometry.

A new porphyrin, peroxyacetic acid uroporphyrin I, has been isolated from the urine of patients with congenital erythropoietic porphyria by reversed phase high performance liquid chromatography. The porphyrin was characterized by high resolution mass spectrometry and by typical chemical reactions of a peroxyacid.

Biosynthesis of vitamin B12: isolation of precorrin-6x, a metal-free precursor of the corrin macrocycle retaining five S-adenosylmethionine-derived peripheral methyl groups.

delta-Aminolevulinic acid and trimethylisobacteriochlorin are converted by cell-free protein preparations from Pseudomonas denitrificans into a metal-free pigment, precorrin-6x. This pigment, which accumulates when the cell-free system lacks NADPH, can be enzymically converted in high yield (greater than 50%) into hydrogenobyrinic acid by the complete enzyme preparation. Double-labeling experiments establish that precorrin-6x carries five C-methyl groups, which appear at C-1, C-2, C-7, C-12 alpha, and C-17 of the hydrogenobyrinic acid formed enzymically from precorrin-6x. This precursor of the corrin macrocycle is at the dehydrocorrin level of oxidation, has undergone ring contraction and extrusion of C-20, but still carries the acetic acid side chain at C-12. It is demonstrated that the conversion of precorrin-6x into hydrogenobyrinic acid specifically requires an NADPH-dependent reduction step.

Separation of porphyrin isomers by high-performance liquid chromatography.

A high-speed reversed-phase high-performance liquid chromatographic method using an octadecylsilyl 3 cm long (3 microns particle size) column to separate the free acids of uroporphyrins I and III and coproporphyrins I and III from each other, and from the type I isomers of several other porphyrin carboxylic acids, is described. Separation of the porphyrins was achieved in less than 8 min, and injections were possible every 12 min. The detection limits of uroporphyrin, coproporphyrin, and mesoporphyrin were 75, 45, and 35 fmol (at a signal-to-noise ratio of 2), respectively. Application of the method to the determination of urinary and liver porphyrin patterns is shown.

Extraction and isolation by high performance liquid chromatography of uroporphyrin and coproporphyrin isomers from biological tissues.

A simple, rapid procedure has been developed for extraction of uroporphyrin and coproporphyrin isomers from biological tissues. The recoveries of known standards of uroporphyrin I and III and coproporphyrin I and III were performed from liver, kidney, testis, and bone marrow of the rat. The extracted samples were analyzed by high performance liquid chromatography. This method is suitable for the study of drug- and toxicant-induced porphyrias characterized by alterations of the ratios of the I and III isomers of uroporphyrin and coproporphyrin.

Analysis and separation of natural and synthetic mixtures of uroporphyrins by high-pressure liquid chromatography.

A new method for the quantitative analysis of mixtures of the methyl esters of uroporphyrins I and III was developed; this can be applied both to the analysis of naturally occurring uroporphyrins and also to their semi-preparative isolation.