Formation Of Lumichrome Research Articles

Divalent anion catalyzed photodegradation of riboflavin: A kinetic study

A study of the effect of some divalent anions (carbonate, oxalate, phthalate, phosphate) on the kinetics of photodegradation reactions of riboflavin (RF) and its pathways, product composition and fluorescence quenching in the pH range 6.0–8.0 has been conducted. The photodegradation of RF in the presence of 0.2–1.0 M divalent anions on visible irradiation leads to the formation of cyclodehydroriboflavin (CDRF) by photoaddition pathway and lumichrome (LC) by photoreduction pathway as the major end products. These products are formed by simultaneous first–order reactions. The divalent anions deviate the photoreduction pathway of RF in favor of photoaddition pathway to yield CDRF. The extent of this variation depends on the strength of RF–divalent anion complex as indicated by an increase in the loss of RF fluorescence due to complex formation. The first– and second–order rate constants for the photodegradation of RF have been determined and the effect of pH on the rates of these reactions evaluated. The rate of divalent anion catalyzed formation of CDRF is maximum at pH 7 and occurs in the order: carbonate > phosphate > oxalate > phthalate. The catalytic activity of the divalent anions increases with the strength of RF–divalent anion complex resulting in an increase in the rate of formation of CDRF and a decrease in the rate of formation of LC by different pathways.

Development of a HPLC method for the simultaneous analysis of riboflavin and other flavin compounds in liquid milk and milk products

A high-performance liquid chromatography method with fluorescence detection was developed for the quantification of riboflavin (RF), flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD) and their photodegradation products, lumichrome (LC) and lumiflavin (LF), in liquid milk and milk products. Both sample preparation and chromatographic separation were studied to avoid acidic conditions that proved to affect flavin stability and degrade FAD into FMN. The sample preparation includes centrifugal skimming and ultrafiltration steps and is suitable for routine application. Linear response was obtained for individual flavins in the respective concentration ranges of interest and relative standard deviation (RSD) was lower than 5%, except for FAD (RSD 11%). The recovery ranged between 80–100%. The proposed method proved to be suitable for assessing flavins in commercial liquid milk and fermented milk products, and for monitoring the degradation of FAD, FMN and RF and the formation of LF and LC in bottled milk exposed to light during shelf storage.

Metal ion mediated photolysis reactions of riboflavin: A kinetic study

The effect of metal ion complexation on the photolysis of riboflavin (RF) using various metal ions (Ag+, Ni2+, Co2+, Fe2+, Ca2+, Cd2+, Cu2+, Mn2+, Pb2+, Mg2+, Zn2+, Fe3+) has been studied. Ultraviolet and visible spectral and fluorimetric evidence has been obtained to confirm the formation of metal-RF complexes. The kinetics of photolysis of RF in metal-RF complexes at pH7.0 has been evaluated. The apparent first-order rate constant (kobs) for the photolysis of RF and the formation of lumichrome (LC) and lumiflavin (LF) (0.001M phosphate buffer) and LC, LF and cyclodehydroriboflavin (CDRF) (0.2–0.4M phosphate buffer) have been determined. The values of kobs indicate that the rate of photolysis of RF is promoted by divalent and trivalent metal ions. The second-order rate constants (kʹ ) for the interaction of metal ions with RF are in the order: Zn2+>Mg2+>Pb2+>Mn2+>Cu2+>Cd2+>Fe2+>Ca2+>Fe3+>Co2+>Ni2+>Ag+. In phosphate buffer (0.2–0.4M), an increase in the metal ion concentration leads to a decrease in the formation of LC compared to that of CDRF by different pathways. The photoproducts of RF have been identified and RF and the photoproducts have simultaneously been assayed by a multicomponent spectrometric method. The mode of photolysis of RF in metal-RF complexes has been discussed.

Effect of phosphate buffer on the complexation and photochemical interaction of riboflavin and caffeine in aqueous solution: A kinetic study

A study of the photodegradation of 5 × 10−5 M riboflavin (RF) in 0.2–1.0 M phosphate buffer in the presence and absence of 2.50 × 10−4 M caffeine at pH 6.0–8.0 has been carried out. RF in phosphate buffer is photodegraded simultaneously by normal photolysis (photoreduction) and photoaddition reactions giving rise to lumichrome (LC) and cyclodehydroriboflavin (CDRF) as the main final products, respectively. RF and its photoproducts, formylmethylflavin (FMF), lumiflavin (LF), LC and CDRF in degraded solution have been determined by a specific multicomponent spectrophotometric method with an accuracy of ±5%. The apparent first-order rate constants for the photodegradation of RF and for the formation of LC and CDRF are 5.47–15.05 × 10−3 min−1, 1.06–8.30 × 10−3 min−1 and 4.31–8.05 × 10−3 min−1, respectively. An increase in phosphate concentration leads to an increase in the rate of formation of CDRF and alters the photodegradation of RF in favor of the photoaddition reaction. This photoaddition reaction is further enhanced in the presence of caffeine which results in a further decrease of the fluorescence of RF in phosphate buffer. Caffeine may facilitate the photoaddition reaction by suppression of the photoreduction pathway of RF.

Effect of borate buffer on the photolysis of riboflavin in aqueous solution

The photolysis of riboflavin (RF) in the presence of borate buffer (0.1–0.5 M) at pH 8.0–10.5 has been studied using a specific multicomponent spectrophotometric method for the determination of RF and photoproducts, formylmethylflavin (FMF), lumichrome (LC) and lumiflavin (LF). The overall first-order rate constants for the photolysis of RF (1.55–4.36 × 10 −2 min −1) and the rate constants for the formation of FMF (1.16–3.52 × 10 −2 min −1) and LC (0.24–0.84 × 10 −2 min −1) have been determined. The values of all these rate constants decrease with an increase in buffer concentration suggesting the inhibition of photolysis reaction by borate species. The kinetic data support the formation of a RF-borate complex involving the ribityl side chain to cause the inhibition of photolysis. The second-order rate constants for the borate inhibited reaction range from 1.17–3.94 × 10 −2 M −1 min −1. The log k-pH profiles for the reaction at various buffer concentrations indicate a gradual increase in rate, with pH, up to 10 followed by a decrease in rate at pH 10.5 probably due to ionization of RF and quenching of fluorescence by borate species. A graph of second-order rate constants against pH is a sigmoid curve showing that the rate of photolysis increases with an increase in pH. The results suggest the involvement of excited singlet state, in addition to excited triplet state, in the formation of LC.

Study on electron transfer of fluorescent probe lumichrome and nucleic acid by laser flash photolysis

Laser flash photolysis (LFP) of lumichrome (LC) has been studied upon laser excitation at 355 nm. The transient absorption spectra of LC in aqueous solution displayed two bands at 380 nm and 540–700 nm for the triplet 3LC, and a wide band at 430–530 nm for the radical anion LC.. The triplet-state lifetime was measured up to 12 µs. It was found that the self-quenching of 3LC by the ground state of LC occurred to produce LC. during LFP. The transient spectra of LC in the presence of electron-donating triphenylamine (TPA), nucleosides and nucleic acids showed that the absorbance of 3LC was strongly quenched by these substrates and the formation of LC. was enhanced in a great extent simultaneously. The bimolecular quenching kinetics were studied and the rate constants of 3LC by a series of quenchers, including TPA, LC, nucleosides and nucleic acids were determined to be 108–109 M−1 s−1. The photo-induced electron-transfer mechanism for these processes involving the triplet 3LC was proposed.

Photodynamics of the small BLUF protein BlrB from Rhodobacter sphaeroides

The BLUF protein BlrB from the non-sulphur anoxyphototrophic purple bacterium Rhodobacter sphaeroides is characterized by absorption and emission spectroscopy. BlrB expressed from E. coli binding FAD, FMN, and riboflavin (called BrlB I) and recombinant BlrB containing only FAD (called BlrB II) are investigated. The dark-adapted proteins exist in two different receptor conformations (receptor states) with different sub-nanosecond fluorescence lifetimes (BLUF r,f and BLUF r,sl). Some of the flavin-cofactor (ca. 8%) is unbound in thermodynamic equilibrium with the bound cofactor. The two receptor conformations are transformed to putative signalling states (BLUF s,f and BLUF s,sl) of decreased fluorescence efficiency and shortened fluorescence lifetime by blue-light excitation. In the dark at room temperature both signalling states recover back to the initial receptor states with a time constant of about 2 s. Quantum yields of signalling state formation of about 90% for BlrB II and about 40% for BlrB I were determined by intensity dependent transmission measurements. Extended blue-light excitation causes unbound flavin degradation (formation of lumichrome and lumiflavin-derivatives) and bound cofactor conversion to the semiquinone form. The flavin-semiquinone further reduces and the reduced flavin re-oxidizes back in the dark. A photo-dynamics scheme is presented and relevant quantum efficiencies and time constants are determined.

Photosensitizing Effect of Riboflavin, Lumiflavin, and Lumichrome on the Generation of Volatiles in Soy Milk

Lumichrome and lumiflavin were formed from riboflavin under light. pH had a significant influence on the formation of lumichrome and lumiflavin from riboflavin. Lumichrome was the only major product from riboflavin under neutral or acidic pH values. Lumiflavin was also formed from riboflavin in basic pH. The maximum concentration of lumiflavin from 100 microM riboflavin at pH 8.5 was 30.9 microM, and it was reached after 2 h of exposure at 1500 lux. The maximum concentration of lumichrome formed from 100 microM riboflavin at pH 4.5, 6.5, or 8.5 was 79.9, 58.7, and 73.1 microM, respectively, after 8, 6, or 2 h of light exposure. The formation of lumichrome and lumiflavin from riboflavin was due to the type I mechanism of the riboflavin photosensitized reaction. Singlet oxygen was also involved in the photosensitized degradation of lumiflavin and lumichrome. The reaction rates of riboflavin, lumiflavin, and lumichrome with singlet oxygen were 9.66 x 10(8), 8.58 x 10(8), and 8.21 x 10(8) M(-1) s(-1), respectively. The headspace oxygen depletion and headspace volatile formation were significant in soy milk containing lumichrome or lumiflavin under light (p < 0.05) and were insignificant (p > 0.05) in the dark. Ascorbic acid could inhibit the total volatile changes of soy milk under light. Soy milk should be protected from light to prevent the photodegradation of riboflavin and the oxidation of soy milk.

Effect of phosphate buffer on photodegradation reactions of riboflavin in aqueous solution

The effect of phosphate buffer on aerobic photodegradation reactions of riboflavin (RF) at pH 7.0 has been studied. The photoproducts of the two major reactions, viz., intramolecular photoreduction and intramolecular photoaddition, have been determined by a specific multicomponent spectrophotometric method. The overall photodegradation of riboflavin in the presence of phosphate buffer involves the participation of both H 2 PO 4 - and HPO 4 2 - species. The second-order rate constants for the H 2 PO 4 - -catalysed photodegradation of riboflavin (normal photolysis) to lumichrome (LC) and HPO 4 2 - -catalysed photodegradation of riboflavin (photoaddition) to cyclodehydroriboflavin (CDRF) are 0.93 × 10 −4 and 4.0 × 10 −4 M −1 s −1, respectively. The addition of 0.25–2.00 M phosphate to RF solutions at pH 7.0 gives rise to RF – HPO 4 2 - complex and hence the quenching of 4–36% fluorescence, respectively. This results in the suppression of normal photolysis leading to the formation of LC in favour of photoaddition to yield CDRF. The present study shows the involvement of H 2 PO 4 - anions in the base-catalysed degradation of riboflavin by normal photolysis vis-a-vis the involvement of HPO 4 2 - anions in photoaddition reactions of riboflavin suggested earlier [M. Schuman Jorns, G. Schollnhammer, P. Hemmerich, Intramolecular addition of the riboflavin side chain. Anion-catalysed neutral photochemistry, Eur. J. Biochem. 57 (1975) 35–48].

A study of simultaneous photolysis and photoaddition reactions of riboflavin in aqueous solution

The photodegradation reactions of riboflavin (RF) in the presence of 0.05–2.00 M phosphate (pH 7.0) have been studied using a specific multicomponent spectrophotometric method. The reactions involve simultaneous photolysis (intramolecular photoreduction) and photoaddition (intramolecular photoaddition) leading to lumichrome (LC) and cyclodehydroriboflavin (CDRF), respectively, as major products. The contribution of each reaction in the overall photodegradation depends upon the phosphate concentration, i.e., higher the phosphate concentration higher the extent of photoaddition. The apparent first-order rate constants for the photodegradation of RF and for the formation of LC and CDRF at 0.25–2.00 M phosphate concentration range from 0.65 to 3.03 × 10 −2, and from 0.41 to 0.99 × 10 −2 and 0.12 to 1.63 × 10 −2 min −1, respectively. The second-order rate constants for the phosphate catalysed photodegradation of RF and for the formation of LC and CDRF are 2.12 × 10 −4 and 0.61 × 10 −4 and 1.41 × 10 −4 M −1 s −1, respectively. Since the formation of CDRF by photoaddition is catalysed by HPO 4 2− ions [Eur. J. Biochem. 57 (1975) 35], it is suggested that H 2PO 4 − ions may be involved in the formation of LC by photolysis. Thus, both H 2PO 4 − and HPO 4 2− ions may catalyse the two major reaction pathways of riboflavin photodegradation, respectively.

A study of simultaneous photolysis and photoaddition reactions of riboflavin in aqueous solution

The photodegradation reactions of riboflavin (RF) in the presence of 0.05–2.00 M phosphate (pH 7.0) have been studied using a specific multicomponent spectrophotometric method. The reactions involve simultaneous photolysis (intramolecular photoreduction) and photoaddition (intramolecular photoaddition) leading to lumichrome (LC) and cyclodehydroriboflavin (CDRF), respectively, as major products. The contribution of each reaction in the overall photodegradation depends upon the phosphate concentration, i.e., higher the phosphate concentration higher the extent of photoaddition. The apparent first-order rate constants for the photodegradation of RF and for the formation of LC and CDRF at 0.25–2.00 M phosphate concentration range from 0.65 to 3.03 × 10 −2 , and from 0.41 to 0.99 × 10 −2 and 0.12 to 1.63 × 10 −2 min −1 , respectively. The second-order rate constants for the phosphate catalysed photodegradation of RF and for the formation of LC and CDRF are 2.12 × 10 −4 and 0.61 × 10 −4 and 1.41 × 10 −4 M −1 s −1 , respectively. Since the formation of CDRF by photoaddition is catalysed by HPO 4 2− ions [Eur. J. Biochem. 57 (1975) 35], it is suggested that H 2 PO 4 − ions may be involved in the formation of LC by photolysis. Thus, both H 2 PO 4 − and HPO 4 2− ions may catalyse the two major reaction pathways of riboflavin photodegradation, respectively.