The preparation and characterization of the diaquo- forms of several incomplete corrinoids: cobyric acid, cobinamide, and three isomeric cobinic acid pentaamides.

The preparation of the diaquo- forms of the incomplete corrinoids, cobyric acid, cobinamide, and three isomeric cobinic acid pentaamides, from the corresponding aquocyanocorrinoids has been accomplished. The compounds were characterized by electronic absorption spectra, pka values, and HPLC/TLC behavior. Upon titration, the acids exhibited three pKa values, but only the pKa corresponding to the carboxylic acid involved the transfer of a full equivalent of protons. In addition, the progress of diaquo- to dihydroxo-titration, and the spectrum of the final products indicated a complex process. These diaquocorrinoids appear to be unstable, and should be stored in solution, frozen, under an inert atmosphere.

Separation and study of corrinoid cobalt-ligand isomers by high-performance liquid chromatography.

Vitamin B12 belongs to a group of complex organo-cobalt compounds, the corrinoids. "Complete" corrinoids, such as B12, contain a nucleotide as the cobalt alpha-(lower)ligand. This nucleotide is also connected to the periphery of the corrin ring. The "incomplete" corrinoids, in contrast, contain simple cobalt alpha-ligands, such as water or cyanide. Using analytical reversed-phase and anion-exchange high-performance liquid chromatography (HPLC), we have been able to study the behavior of several aquocyano-"incomplete" corrinoids: three isomeric cobinic acid pentaamides, cobinamide, and cobyric acid, all of which exist as thermally unstable isomers, which separate during HPLC. All but one of these corrinoids gave isomer mixtures of 1:1, the exception giving mixtures of 2:1 to 3:1. The separated stereoisomers had different retention times and were collected from analytical columns for further study.

High-performance liquid chromatographic analysis of the (cyanoaquo) stereoisomers of several putative vitamin B12 precursors.

The cyanoaquo and aquocyano stereoisomers of several putative vitamin B12 precursors are reversibly formed and can be separated using analytical high-performance liquid chromatographic methods. The behavior of these stereoisomers varies somewhat depending on the type of column used and the chromatographic conditions employed. Both reversed-phase and ion-exchange columns can be used to observe the reversible formation and separation of the stereoisomers of (H2O,CN)cobyric acid, cobinamide and the cobinic acid pentaamide-1, -2 and -3 structural isomers. The greatest differences in retention times are seen when the pH of the eluting buffer is less than 4.0 and the buffer contains no KCN.

Amidation of, and (R)-1-amino-2-propanol attachment to, the corrin ring during vitamin B-12 biosynthesis by Clostridium tetanomorphum extracts.

Two intermediate stages in cobalamin biosynthesis, amidation of carboxylic acid groups in the corrin ring and (R)-1-amino-2-propanol attachment at propionic acid position f, have been studied using cell-free extracts from the obligate anaerobe Clostridium tetanomorphum. The preparation of an incomplete corrinoid, probably cobinic acid-a,c,d,e,g-pentaamide, as an in vitro amidation substrate was accomplished via mild acid hydrolysis of cobinamide. Weak, but reproducible activities for both amidation and (R)-1-amino-2-propanol attachment were found in crude, nucleic acid-free and DE-52 column-purified protein fractions. The amidation reaction was glutamine-dependent in crude fractions, but became ammonium ion-dependent in more purified fractions. Significant problems encountered were (a) the weak and unstable character of both enzyme activities, and (b) the irreversible changes in the visible spectra of the incomplete corrinoids employed as substrates caused by use of thiol-reducing agents in the buffers and assays.

Formation of delta-aminolevulinic acid from glutamic acid by a partially purified enzymes system from wheat leaves.

A method for partial purification of an enzyme system from greening wheat leaves which converts 14C-labeled glutamate to delta-aminolevulinic acid is described. The purification entails the successive use of anion and cation exchange, followed by molecular sieving. The enzyme system is unstable in crude form, but the stability is markedly increased after column chromatography on CM-cellulose. The pH profile and the cofactor requirements suggest that at least two enzymes are involved.

Vitamin B-12 biosynthesis. A model system for isopropanolamine formation by reaction between reduced corrinoid and threonine.

When a solution of 14C-labeled L-threonine is incubated with aquocobalamin in the presence of excess of a reductant such as 2-mercaptoethanol or sodium borohydride, a labeled corrinoid adduct is formed. The corrinoid participates stoichiometrically, and not catalytically. Since 35S-labeled 2-mercaptoethanol is not part of the adduct, the reduction of the corrinoid from the Co(III) to the Co(II) form is a prerequisite for the reaction. Various independent tests show the formation of isopropanolamine. The reaction system is an attractive model for the so far unexplained decarboxylation of threonine to isopropanolamine in the course of corrinoid biosynthesis.

Simultaneously obtained skin-puncture serum, skin-puncture plasma, and venous serum compared, and effects of warming the skin before puncture.

Specimens of skin-puncture serum and plasma and venous serum were simultaneously obtained from healthy adults and in each specimen the concentrations of 12 chemical constituents were measured. No clinically important difference was found in the concentrations of the constituents measured in skin-puncture serum and plasma with or without warming the skin before puncture. When the concentration of each of the measured constituents was compared in skin-puncture specimens and venous serum there were important differences in the concentrations of glucose, potassium, total protein, and calcium. Except for glucose, the concentrations of these were higher in venous serum. The degree of hemolysis was the same in skin-puncture serum and plasma, but less in venous serum. The greater hemolysis in skin-puncture specimens was not reflected in a clinically important increase in the values of lactate dehydrogenase and potassium. We conclude that there is a difference in the concentration of some chemical constituents in skin-puncture specimens and venous serum and that these differences should be considered when results for these types of specimens are compared.