Cobalamin's (Vitamin B12) Surprising Function as a Photoreceptor.

Cobalamin (Vitamin B12) is an adenosyl- or methyl-donating cofactor for many enzymes, yet many proteins with unknown or nonenzymatic function also contain B12-binding domains. Recent studies show that light excitation energy can promote covalent linkage of B12 to transcription factors with this linkage, affecting gene expression. Thus, B12 now has a newly described regulatory function. Here, our bioinformatics analysis reveals other transcription factors, photoreceptors, kinases, and oxygen sensors that harbor a B12-binding domain that could also regulate activity in response to light absorption.

Vitamin B12: a tunable, long wavelength, light-responsive platform for launching therapeutic agents.

Light-responsive agents offer the promise of targeted therapy, whose benefits include (i) prolonged action at the target site, (ii) overall reduced systemic dosage, (iii) reduced adverse effects, and (iv) localized delivery of multiple agents. Although photoactivated prodrugs have been reported, these species generally require short wavelengths (<450 nm) for activation. However, maximal tissue penetrance by light occurs within the "optical window of tissue" (600-900 nm), well beyond the wavelength range of most existing photocleavable functional groups. Furthermore, since multidrug therapy holds promise for the treatment of complex diseases, from cancer to neurological disorders, controlling the action of multiple drugs via wavelength modulation would take advantage of a property that is unique to light. However, discrimination between existing photoresponsive moieties has thus far proven to be limited. We have developed a vitamin B12/light-facilitated strategy for controlling drug action using red, far-red, and NIR light. The technology is based on a light-triggered reaction displayed by a subset of B12 derivatives: alkyl-cob(III)alamins suffer photohomolysis of the C-Co(III) bond. The C-Co(III) bond is weak (<30 kcal/mol), and therefore all wavelengths absorbed by the corrin ring (330-580 nm) induce photocleavage. In addition, by appending fluorophores to the corrin ring, long wavelength light (>600 nm) is readily captured and used to separate the Co-appended ligand (e.g., a drug) from B12. Consequently, it is now feasible to preassign the wavelength of homolysis by simply installing a fluorescent antenna with the desired photophysical properties. The wavelength malleability inherent within this strategy has been used to construct photoresponsive compounds that launch different drugs by simply modulating the wavelength of illumination. In addition, these phototherapeutics have been installed on the surface and interior of cells, such as erythrocytes or neural stem cells, and released upon expoure to the appropriate wavelength. We have shown that cytotoxic agents, such as doxorubicin, anti-inflammatories, such as dexamethasone, and anti- and pro-vascular agents are readily released from cellular vehicles as biologically active agents. We have also demonstrated that the concept of "optical window of tissue" phototherapeutics is not just limited to prodrugs. For example, stem cells have received considerable attention in the area of regenerative medicine. Hydrogels serve as scaffolds for stem cell growth and differentiation. We have shown that the formation of hydrogels can be triggered, in the presence of cells, using appropriately designed alkyl-cob(III)alamins and long wavelength light. The potential applications of phototherapeutics are broad and include drug delivery for a variety of indications, tissue engineering, and surgery.

Structural basis for gene regulation by a B12-dependent photoreceptor.

Photoreceptor proteins enable organisms to sense and respond to light. The newly discovered CarH-type photoreceptors use a vitamin B12 derivative, adenosylcobalamin, as the light-sensing chromophore to mediate light-dependent gene regulation. Here we present crystal structures of Thermus thermophilus CarH in all three relevant states: in the dark, both free and bound to operator DNA, and after light exposure. These structures provide visualizations of how adenosylcobalamin mediates CarH tetramer formation in the dark, how this tetramer binds to the promoter -35 element to repress transcription, and how light exposure leads to a large-scale conformational change that activates transcription. In addition to the remarkable functional repurposing of adenosylcobalamin from an enzyme cofactor to a light sensor, we find that nature also repurposed two independent protein modules in assembling CarH. These results expand the biological role of vitamin B12 and provide fundamental insight into a new mode of light-dependent gene regulation.

Excited state electron transfer after visible light absorption by the Co(I) state of vitamin B12.

The first example of excited state electron transfer from cob(I)alamin is reported herein. Vitamin B12 was anchored to a mesoporous TiO2 thin film and electrochemically reduced to the cob(I)alamin form. Pulsed laser excitation resulted in rapid excited state electron transfer, ket > 10(8) s(-1), followed by microsecond interfacial charge recombination to re-form cob(I)alamin. The supernucleophilic cob(I)alamin was found to be a potent photoreductant. The yield of excited state electron transfer was found to be excitation wavelength dependent. The implications of this dependence are discussed.

Cyanocobalamin solutions as potential dosimeters in low-dose food irradiations.

Potential of aqueous solutions of cyanocobalamin in gamma radiation dosimetry was investigated. The solutions are inexpensive, nontoxic and easy-to-prepare dosimeters, which could be useful for measuring gamma radiation doses in various applications, such as quarantine treatment of fruit or insect disinfestation of grains and pulses. The optical absorbance of cyanocobalamin solutions of the optimal concentration 0.08 mM decreases with increasing radiation dose. The reproducible dependence of the absorbance decrease on the dose can be described with a polynomial. Pre- and post-irradiation stability of the solution absorbance, as well as effects of the irradiation temperature and dose rate, were studied. The response is not significantly affected by storage of the irradiated dosimeters under ambient conditions for 20 days. The performance characteristics of this chemical dosimetry system suggest that it can be useful to measure doses in irradiations of food.

Heat shock transcriptional responses in an MC-Producing Cyanobacterium (Planktothrix agardhii) and its MC-deficient mutant under high light conditions.

Microcystins (MCs) are the most commonly-reported hepatotoxins produced by various cyanobacterial taxa in fresh waters to constitute a potential threat to human and animal health. The biological role of MCs in the producer organisms is not known, and it would be very useful to understand the driving force behind the toxin production. Recent studies have suggested that MCs may have a protective function in cells facing environmental stress. Following this starting premise, we speculate that under adverse conditions the expression of stress-related genes coding for Heat Shock Proteins (Hsp) might be different in an MC-producing strain and its MC-deficient mutant. We therefore used RT-qPCR to compare the expression of 13 hsp genes of an MC-producing strain of Planktothrix agardhii (CYA126/8) and its MC-deficient ΔmcyD mutant over different periods of exposure to high light stress (HL). Three reference genes (RGs) were selected from six candidates to normalize the RT-qPCR data. Of these three RGs (rsh, rpoD, and gltA), gltA is used here for the first time as an RG in prokaryotes. Under HL stress, five genes were found to be strongly up-regulated in both strains (htpG, dnaK, hspA, groES, and groEL). Unexpectedly, we found that the MC-producing wild type strain accumulated higher levels of htpG and dnaK transcripts in response to HL stress than the MC-deficient mutant. In addition, a significant increase in the mcyE transcript was detected in the mutant, suggesting that MCs are required under HL conditions. We discuss several possible roles of MCs in the response to HL stress through their possible involvement in the protective mechanisms of the cells.

Photodegradation of cobalamins in aqueous solutions and in human blood.

Vitamin B12 (cobalamin) is required for proper red blood cell formation, neurologic function, and DNA synthesis. Cobalamins in solutions are light sensitive, but no comprehensive study has been performed to compare the photostability of different cobalamins under UVA exposure. Their indirect photodegradation due to their antioxidant properties and their photostability in vivo have also not been studied so far. The photodegradation of four cobalamins (methylcobalamin (MeCbl), adenosylcobalamin (AdCbl), hydroxocobalamin (OHCbl) and cyanocobalamin (CNCbl)) under UVA exposure in aqueous solutions (pH=7.4) have been investigated by absorption spectroscopy. The photodegradation of OHCbl in the absence and presence of the endogenous photosensitizer riboflavin was studied. Serum vitamin B12 concentrations before and after summer were measured in four patients with psoriasis. All studied cobalamins are photolabile. The biologically active forms of cobalamin, AdCbl and MeCbl, are converted to OHCbl within seconds during UVA exposure. OHCbl is the most stable cobalamin. However, reactive oxygen species increases the degradation rate of OHCbl. Our pilot study on humans demonstrates that serum vitamin B12 concentrations are not significantly affected during summertime in Norway. Further work is needed to determine vitamin B12 photostability in humans living at lower latitudes or using sunbeds.

Use of viscosigens to stabilize vitamin B12 solutions against photolysis.

Glycerol stabilizes cyanocob(III)alamin (vitamin B12) against anaerobic photolysis by a high-intensity UV light source or a low-intensity fluorescent light source. The likely mechanism for stabilization by viscosigens is a decrease in diffusion and an enhancement of radical-pair recombination. Glycerol (25% or greater) stabilizes therapeutic doses of vitamin B12 in single-dose Tubex injection vials against adventitious photolysis by fluorescent light.

[Stability of folic acid and vitamin B12 in TPN]. / Estabilidad del ácido fólico y vitamina B12 en NPT.

The stability of folic acid (FA) in mixtures of Total Parenteral Nutrition has been and is a controversial subject, with discussion concerning the influence of factors such as temperature, light and storage time. As regards the stability of the vitamin B12, there are few studies in scientific literature. For all those reasons, we consider it necessary to make a proper study to evaluate the influence of different factors in the stability of both vitamins. The study was made on 3 liter TPN bags of the EVA type, the composition of which was as follows: AA (85g), glucosa (225g), fat (50g), Na (86mEq), K (60 mEq), Ca (15 mEq), Cl (90 mEq), P (17 mmol) acetate (149 mEq) and 10 ml of MVI-12 which contain 400 micrograms of PA and 5 micrograms of Vitamin B 12. Consideration was also given to the stability of these two vitamins in the same diet, to which were added 10 ml of a commercial preparation of oligo-elements. Six TPN bags were prepared (without oligo-elements); two of them were kept in a fridge and protected from the light, two were kept at room temperature and protected from the light and the other two at room temperature without protection from the light. Samples were taken from all the bags immediately after their preparation and after 24, 48, 72 and 96 hours. The same process was carried with other TPN bags which did contain oligo-elements. The method for determining FA and Vitamin B12 was by radioassay.(ABSTRACT TRUNCATED AT 250 WORDS)

High-performance liquid chromatographic separation of cobalamins.

Physiological cobalamins were separated by means of high-performance liquid chromatography (HPLC). Optimal conditions for elution of methylcobalamin, adenosylcobalamin, hydroxycobalamin and cyanocobalamin were determined. Excellent separation and resolution of these physiological cobalamins by HPLC were achieved. In addition, several cobalamin analogues were also studied and shown to be separable from the physiological forms. HPLC provides a rapid, sensitive, reproducible means of characterizing physiological cobalamins.

Effect of X-irradiation on vitamin B12 binding capacity to intrinsic factor.

The effects of whole-body X-irradiation on vitamin B12-protein complex formation in gastric juice after oral administration of [57Co]-B12 have been studied. Two proteins with B12-binding activity have been isolated by gel filtration from gastric juice. 57Co-activity, recovered from B12-protein complex in gastric juice, is found to be about 30% less in the X-irradiated rat. In serum, vitamin B12 is mainly associated with alpha1-globulin. Radioactivity distribution in serum globulins after intraperitoneal injection of [57Co]-B12 was similar in control and X-irradiated rats.