Rapid analysis of cobalamin coenzymes and related corrinoid analogs by high-performance liquid chromatography

Cobalamin coenzymes and a series of related corrinoid analogs have been analyzed by high-performance liquid chromatography on reverse-phase C₈ and C₁₈ columns using both isocratic and gradient elution systems and 254-nm absorbance detection. In the isocratic mode, retention times for sulfitocobalamin, cyanocobalamin, methylcobalamin, adenosylcobalamin, and aquacobalamin on a LiChrosorb C₈ column were 1.1, 1.6, 2.2, 2.9, and 4.7 min, respectively. In the gradient mode, corresponding retention times were 9.7, 10.2, 12.8, 11.4, and 9.0 min. Closely related structural analogs of adenosylcobalamin such as 1,N⁶-ethenoadenosylcobalamin, formycinylcobalamin, and 2,6-diaminonebularinylcobalamin were clearly resolved from naturally occurring cobalamins by isocratic elution. In the gradient system, the order of elution of cobalamins was related to the hydrophobicity of the upper-axial ligand. This was demonstrated by determining the retention times of aminoalkylcobalamin homologs (C₂, 9.8; C₃, 10.5; C₅, 11.2; C₈, 12.8; and C₁₁, 14.9 min). The usefulness of this method was demonstrated by analyzing ⁵⁷Co-labeled cobalamins present in extracts of Lactobacillus leichmannii and murine leukemia L1210 cells.     

Complete disulfide bond assignment of a recombinant immunoglobulin G4 monoclonal antibody

Surface plasmon resonance biosensor analysis was used to evaluate the thermodynamics and binding kinetics of naturally occurring and synthetic cobalamins interacting with vitamin B(12) binding proteins. Cyanocobalamin-b-(5-aminopentylamide) was immobilized on a biosensor chip surface to determine the affinity of different cobalamins for transcobalamin, intrinsic factor, and nonintrinsic factor. A solution competition binding assay, in which a surface immobilized cobalamin analog competes with analyte cobalamin for B(12) protein binding, shows that only recombinant human transcobalamin is sensitive to modification of the corrin ring b-propionamide of cyanocobalamin. A direct binding assay, where recombinant human transcobalamin is conjugated to a biosensor chip, allows kinetic analysis of cobalamin binding. Response data for cyanocobalamin binding to the transcobalamin protein surface were globally fitted to a bimolecular interaction model that includes a term for mass transport. This model yields association and dissociation rate constants of k(a) = 3 x 10(7) M(-1) s(-1) and k(d) = 6 x 10(-4) s(-1), respectively, with an overall dissociation constant of K(D) = 20 pM at 30 degrees C. Transcobalamin binds cyanocobalamin-b-(5-aminopentylamide) with association and dissociation rates that are twofold slower and threefold faster, respectively, than transcobalamin binding to cyanocobalamin. The affinities determined for protein-ligand interaction, using the solution competition and direct binding assays, are comparable, demonstrating that surface plasmon resonance provides a versatile way to study the molecular recognition properties of vitamin B(12) binding proteins.     

An improved enzymatic cycle for nicotinamide-adenine dinucleotide phosphate.

We describe the use of column chromatography on the nonpolar adsorbent. Amberlite XAD-2, and on silanized silica gel in the desalting and partial purification of cobalamins. These techniques are both simpler and more versatile than phenol extraction, without sacrificing efficiency. In addition, a solvent system for thin-layer chromatography on silanized silica gel is described which rapidly separates naturally occurring cobalamins.     

Conversion and Distribution of Cobalamin in Euglena gracilis z, with Special Reference to Its Location and Probable Function within Chloroplasts

Cobalamin is essentially required for growth by Euglena gracilis and shown to be converted to coenzyme forms promptly after feeding cyanocobalamin. Concentrations of coenzymes, methylcobalamin, and 5′-deoxyadenosylcobalamin, reached about 1 femtomole/10⁶ cells 2 hours after feeding cyanocobalamin to cobalamin-limited cells. Cobalamins all were bound to proteins in Euglena cells and located in subcellular fractions of chloroplasts, mitochondria, microsomes, and cytosol. Incorporated cobalamin into chloroplasts was localized in thylakoids. Methylcobalamin existed in chloroplasts, mitochondria, and cytosol, while 5′-deoxyadenosylcobalamin was in mitochondria and the cytosol, 2 h after feeding cyanocobalamin to Euglena cells. Quantitative alterations of methylcobalamin and 5′-deoxyadenosylcobalamin in chloroplasts suggest their important functions as coenzymes in this organelle. The occurrence of functional cobalamins in chloroplasts has not been reported in other photosynthetic eukaryotes.     

Mechanism of reactivation of coenzyme B12-dependent diol dehydratase by a molecular chaperone-like reactivating factor.

The mechanism of reactivation of diol dehydratase by its reactivating factor was investigated in vitro by using enzyme. cyanocobalamin complex as a model for inactivated holoenzyme. The factor mediated the exchange of the enzyme-bound, adenine-lacking cobalamins for free, adenine-containing cobalamins through intermediate formation of apoenzyme. The factor showed extremely low but distinct ATP-hydrolyzing activity. It formed a tight complex with apoenzyme in the presence of ADP but not at all in the presence of ATP. Incubation of the enzyme.cyanocobalamin complex with the reactivating factor in the presence of ADP brought about release of the enzyme-bound cobalamin, leaving the tight apoenzyme-reactivating factor complex. Although the resulting complex was inactive even in the presence of added adenosylcobalamin, it dissociated by incubation with ATP, forming the apoenzyme, which was reconstitutable into active holoenzyme with added coenzyme. Thus, it was established that the reactivation of the inactivated holoenzyme by the factor in the presence of ATP and Mg2+ takes place in two steps: ADP-dependent cobalamin release and ATP-dependent dissociation of the apoenzyme.factor complex. ATP plays dual roles as a precursor of ADP in the first step and as an effector to change the factor into the low-affinity form for diol dehydratase. The enzyme-bound adenosylcobalamin was also susceptible to exchange with free adeninylpentylcobalamin, although to a much lesser degree. The mechanism for discrimination of adenine-containing cobalamins from adenine-lacking cobalamins was explained in terms of formation equilibrium constants of the cobalamin.enzyme.reactivating factor ternary complexes. We propose that the reactivating factor is a new type of molecular chaperone that participates in reactivation of the inactivated enzymes.     

A simple, convenient and direct method for assessing the purity of cobalamins.

A straightforward and simple, but powerful and direct, method is presented for both the detection and quantitation of cobalamin impurities in either commercial cobalamins or in metastable cobalamins (Cbls), such as RSCbls. The method is, quite simply, the use of the aromatic region of the 1H NMR of cobalamins; it is a method developed as an outgrowth of our work preparing metastable thiolatocobalamins (RSCbls) and is a method that proved necessary for characterizing those (and by inference other) cobalamins unstable to HPLC separation conditions (i.e., and, therefore, where the normally powerful HPLC method so commonly used in cobalamin chemistry fails). Despite considerable, prior, modern multidimensional NMR literature on cobalamins, the present method has not yet been indicated explicitly, nor has anyone reported previously the NMR data required to prove that the method works (i.e., the data for a series of cobalamins and their common impurities proving that they have different chemical shifts in the aromatic region of their 1H NMR when examined under identical NMR solvent, pH and other conditions). The direct NMR method is easy to perform, readily quantitated and applicable to species unstable to the HPLC conditions required to separate cobalamin impurities. The results have allowed quantitation of the 5-11% impurities in, for example, commercial HOCb1.HX, results which document that some commercially available cobalamins are not as pure as the manufacturers' claims.     

Method for analysis of dopamine sulfate isomers by high performance liquid chromatography

Conjugated dopamine occurs in the tissues and fluids of many species, and much of this is thought to occur as dopamine sulfate. This paper describes the development and use of a method utilizing reversed-phase paired-ion high performance liquid chromatography to separate and quantitate each of the two naturally occurring dopamine sulfate isomers. Use of the method permitted demonstration of dopamine-3-0-sulfate in human urine from drug-free control subjects. It was found that this compound accounted for 73.1 ± 27% of the total daily conjugated dopamine excretion in the four subjects studied.     

Liquid chromatography of pyridine nucleotides and associated compounds and isolation of several analogs of nicotinamide adenine dinucleotide phosphate.

A single-column technique is described for separating the naturally occurring nicotinic acid- and nicotinamide-containing nucleosides and nucleotides. A unique feature of the system is the use of conductivity measurements as a reproducible means of peak identification. The chromatographic system has been used to isolate a number of NADP⁺ analogs and to characterize their enzymatic hydrolysis products. These analogs include the α-anomer of NADP⁺, the nicotinic acid analog of NADP⁺, and a series of NADP⁺ derivatives that contain phosphate in the 3′-position.     

The stereoselectivity of the Clostridium perfringens phospholipase C: hydrolysis of thiophosphate analogs of phosphatidylcholine

Thiophosphate containing analogs of phosphatidylcholine have been synthesized with varying degrees of structural complexity. These analogs have been used in a continuous spectrophotometric assay for phospholipase C from Clostridium perfringens in order to examine the requirement for substrate ester functionalities and the stereoselectivity of the enzyme. The substrate analogs with ester groups in the nonpolar portion of the molecule were acceptable substrates for phospholipase C, while those analogs without ester functionalities were not hydrolyzed. Substrate analogs with chiral centers were resolved using the stereospecificity of phospholipase A2 from Crotalus atrox venom. These resolved substrates were used to study the biphasic hydrolytic time courses observed when rac-dioctanoylphosphatidylthiocholine was used as substrate. The "naturally occurring" enantiomer with R absolute configuration was rapidly hydrolyzed in the presence of phospholipase C while the "nonnaturally occurring" enantiomer with S configuration was slowly hydrolyzed only after a long induction or "lag" period. The selectivity for the R enantiomer over the S enantiomer can be lessened by altering the composition of the substrate micelles resulting in accelerated rates of hydrolysis of the S enantiomer.     

Some Metabolic Effects of Sodium Nitroprusside in Man

The infusion of sodium nitroprusside during surgical operations produced plasma levels of cyanide of up to four times the control value. Plasma thiocyanate showed little change except during prolonged infusion of the drug, but total plasma B₁₂ tended to fall, as did methylcobalamin. Other cobalamins showed little change after nitroprusside infusion for short periods. During longer infusions the ratio of methylcobalamin to adenosyl + hydroxocobalamin fell and cyanocobalamin concentrations were high.     

Tissue distribution of endogenous cobalamins and other corrins in the rat, cat and guinea pig.

1. Methylcobalamin, adenosylcobalamin, hydroxocobalamin and cyanocobalamin have been estimated by a chromato-bioautographic techniques in 16 tissues from healthy rats and in five guinea pig tissues. 2. Plasma and erythrocyte cobalamins have been estimated in rats, cats and guinea pigs and the results compared with those in man. 3. Unidentified corrins were detected in 8 of the 16 rat tissues and in 3 of the 5 guinea pig tissues analysed, but were not present in tissues from specific pathogen-free rats nor in the standard laboratory diet. 4. Adenosylcobalamin was the major corrin in 8 of the 16 rat tissues. In the remainder hydroxocobalamin predominated or was present in equal proportions with adenosylcobalamin. Methylcobalamin was detected in the majority of rat tissues but at levels much lower than those in human tissues. Small amounts of cyanocobalamin were detected also and levels were higher than those of methylcobalamin in 8 of the 16 tissues. 5. In the rat, cat and guinea pig, levels of methylcobalamin and hydroxobalamin were higher in erythrocytes than in plasma, a pattern almost the complete reverse of that in man.     

Naturally occurring cobalamins have antimalarial activity

The acquisition of resistance by malaria parasites towards existing antimalarials has necessitated the development of new chemotherapeutic agents. The effect of vitamin B(12) derivatives on the formation of beta-haematin (synthetic haemozoin) was determined under conditions similar to those in the parasitic food vacuole (using chloroquine, a known inhibitor of haemozoin formation for comparison). Adenosylcobalamin (Ado-cbl), methylcobalamin (CH(3)-cbl) and aquocobalamin (H(2)O-cbl) were approximately forty times more effective inhibitors of beta-haematin formation than chloroquine, cyanocobalamin (CN-cbl) was slightly more inhibitory than chloroquine, while dicyanocobinamide had no effect. It is proposed that the cobalamins exert their inhibitory effect on beta-haematin formation by pi-interactions of their corrin ring with the Fe(III)-protoporphyrin ring and by hydrogen-bonding using their 5,6-dimethylbenzimidazole/ribose/sugar side-chain. The antimalarial activity for the cobalamins (Ado-cbl>CH(3)-cbl>H(2)O-cbl>CN-cbl) was found to be less than that for chloroquine or quinine. Ado-cbl, CH(3)-cbl and CN-cbl do not accumulate in the parasite food vacuole by pH trapping, but H(2)O-cbl does. Unlike humans, the malaria parasite has only one enzyme that uses cobalamin as a cofactor, namely methionine synthase, which is important for growth and metabolism. Thus cobalamins in very small amounts are necessary for Plasmodium falciparum growth but in larger amounts they display antimalarial properties.     

Pyrene-labeled lipids: versatile probes of membrane dynamics in vitro and in living cells

Pyrene-labeled analogs of fatty acids have been studied as probes of lipid metabolism in vitro and in cultured cells. Procedures for the synthesis of complex pyrenyl lipids and the analytical methods for their separation and quantification are described. Pyrenyl-lipids have been used to quantify the relationship between lipid structure and the rates of spontaneous lipid transfer. Modifications of these methods have also been used to monitor protein-mediated lipid transfer, lipolysis and lipid translocation across bilayer membranes. According to several criteria, pyrene dodecanoic acid has been identified as a good analog of some naturally occurring fatty acids. Digital imaging microscopy has been used to monitor the rate of accumulation of pyrenyl lipids in living cells.     

Synthetic procedures for the preparation of deuterium-labeled analogs of naturally occurring steroids

The object of this article is to review the procedures that have been published concerning the preparation of deuterium-labeled analogs of naturally occurring steroid hormones. In combination with mass spectrometric methods, these stable isotope-labeled compounds should be applicable for human metabolism studies or as internal standards. Deuteration techniques for the elucidation of stereochemical problems, procedures for the preparation of monodeuterated steroids, and synthesis of deuterated analogs of nonbiologic steroids have therefore not been included in this review.     

Preparation of Radioactive Labelled (57Co) Sulfitocobalamin

Abstract

Farquharson and Adams (Br. J. Nutr. 36, 127-135 (1976)) have identified sulfitocobalamin (S0₃?Cbl) as one of the naturally occurring cobalamins (Cbls) in foods. We have devised a method of making radioactive labelled S0₃?Cbl for invivo and in vitro studies of this form of Cbl. ⁵⁷Co labelled cyanocobalamin (⁵⁷Co CN-Cbl) was acid photolyzed to ⁵⁷Co hydroxocobalamin (⁵⁷Co OH-Cbl) followed by ligand substitution with S0₃ ^?2 ion from aqueous sodium (meta) bisulfite in the dark. The resulting ⁵⁷Co SO₃?Cbl was purified by organic extraction and cation ex-change chromatography. The final preparation was >99% Co⁵⁷ S0₃?Cbl with an overall yield of >70%, stable for up to four weeks at 20°C in the dark, and capable of binding to the human Cbl binding proteins Transcobalamin II (TC II), Intrinsic factor (IF) and Salivary R. This method allows a simple 1 day preparation of high specific activity labelled ⁵⁷Co S0₃?Cbl for biological studies.     

Quantitation of hydroxyproline isomers in acid hydrolysates by high-performance liquid chromatography

A method has been developed to rapidly separate and quantitate levels of hydroxy-L-proline isomers in tissue hydrolysates. The procedure incorporates derivatization of the imino acids with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole chloride followed by separation by high-performance liquid chromatography employing two C18 reverse-phase columns connected in series. Conditions for the derivatization procedure have been optimized for the selective reactivity of imino acids. The derivatized imino acid fractions are then quantitated spectrophotometrically at 495 nm. Using this technique, quantities above 40 pmol are readily detected for trans-4-hydroxyl-L-proline, trans-3-hydroxyl-L-proline, proline, and other imino acid analogs. The method is applicable to a wide range of clinical and experimental tissues.     

Reverse Structural Genomics: AN UNUSUAL FLAVIN-BINDING SITE IN A PUTATIVE PROTEASE FROM BACTEROIDES THETAIOTAOMICRON

The structure of a putative protease from Bacteroides thetaiotaomicron features an unprecedented binding site for flavin mononucleotide. The flavin isoalloxazine ring is sandwiched between two tryptophan residues in the interface of the dimeric protein. We characterized the recombinant protein with regard to its affinity for naturally occurring flavin derivatives and several chemically modified flavin analogs. Dissociation constants were determined by isothermal titration calorimetry. The protein has high affinity to naturally occurring flavin derivatives, such as riboflavin, FMN, and FAD, as well as lumichrome, a photodegradation product of flavins. Similarly, chemically modified flavin analogs showed high affinity to the protein in the nanomolar range. Replacement of the tryptophan by phenylalanine gave rise to much weaker binding, whereas in the tryptophan to alanine variant, flavin binding was abolished. We propose that the protein is an unspecific scavenger of flavin compounds and may serve as a storage protein in vivo.     

Molecular recognition in the binding of vitamin B12 by the cobalamin-specific Intrinsic Factor

Equilibrium constants (given as log K/M-1) have been determined at pH 7.4 and 4 degrees C for binding by porcine Intrinsic Factor (B12-binding protein from the gut, specific for the 'cobalamin' series of Co corrinoids) of vitamin B12 or cyanocobalamin (10.5), cyanocobinamide, alpha-ribazole and alpha-ribazole-phosphate (main fragments produced by cleaving off the 'cobalamin' side-chain, all less than or equal to 3), and cyanocobinamide in the presence of greater than or equal to 10(-9) M ribazole (5.6 and independent of ribazole concentration), i.e. ribazole catalyses the binding of the cobinamide. It is proposed that the specificity of Intrinsic Factor for the cobalamins depends on the presence of the ribazole fragment in the cobalamin side-chain to promote an essential change in conformation before the corrinoid fragment can be bound.     

Artificial analogs of naturally occurring tumor promoters as biochemical tools and therapeutic leads

Tumor promoters are non-carcinogenic chemicals that enhance tumor formation when administered repeatedly after a low dose of a carcinogen. Phorbol esters, teleocidins, and aplysiatoxins are typical examples of naturally occurring tumor promoters. All of them share the ability to bind and activate protein kinase C (PKC) despite the differences in their chemical structures. A variety of analogs with unique chemical and biological properties have been developed to analyze the molecular mechanism of tumor promotion through PKC activation. Moreover, coupled with the emerging significance of PKC in the pathological processes of Alzheimer's disease (AD) and acquired immune deficiency syndrome (AIDS) as well as cancer, several efforts have been made recently to generate analogs of tumor promoters with therapeutic potential. This review focuses on artificial analogs of phorbol esters, teleocidins, and aplysiatoxins, and discusses their potential as biochemical tools and therapeutic leads.     

Artificial Analogs of Naturally Occurring Tumor Promoters as Biochemical Tools and Therapeutic Leads

Tumor promoters are non-carcinogenic chemicals that enhance tumor formation when administered repeatedly after a low dose of a carcinogen. Phorbol esters, teleocidins, and aplysiatoxins are typical examples of naturally occurring tumor promoters. All of them share the ability to bind and activate protein kinase C (PKC) despite the differences in their chemical structures. A variety of analogs with unique chemical and biological properties have been developed to analyze the molecular mechanism of tumor promotion through PKC activation. Moreover, coupled with the emerging significance of PKC in the pathological processes of Alzheimer's disease (AD) and acquired immune deficiency syndrome (AIDS) as well as cancer, several efforts have been made recently to generate analogs of tumor promoters with therapeutic potential. This review focuses on artificial analogs of phorbol esters, teleocidins, and aplysiatoxins, and discusses their potential as biochemical tools and therapeutic leads.