[Synephrine in dietary supplements and specialized foodstuffs: biological activity, safety and methods of analysis].

Synephrine is a natural protoalkaloid of the bitter orange Citrus aurantium L., it has structural similarity to ephedrine and adrenaline. Synephrine in the form of bitter orange extract is widely used as an ingredient of dietary supplements (DS) and specialized foodstuffs (SF) intended for weight loss and fitness improvement. Along with thermogenic and lipolytic effects, synephrine can cause cardiovascular side effects, especially when combined with caffeine and physical activity. This aspect is important, insofar as the main consumers of weight loss products are overweight people who are at risk of developing cardiovascular diseases. The aim of the research is a hygienic assessment of the usage of bitter orange extract and synephrine in DS and SF, which includes an analysis of approaches to technical regulation in the Russian Federation and abroad, a review of data on biological activity, safety, types of adulteration and methods for the determination of citrus protoalkaloids. Results. The adrenergic effect of bitter orange is caused by the presence of R-(-)-psynephrine, making up about 90% or more of the total protoalkaloids. Dry bitter orange fruit extracts, standardized to synephrine content, which can vary from 4 to 98%, are used in the production of DS and SF. Synephrine is a weak adrenergic agonist, acting primarily through ß3-adrenergic receptors, stimulating lipolysis. Because of insufficient safety data, the consumption of synephrine is regulated in the Russian Federation and abroad. The upper permissible level of synephrine consumption in the Russian Federation is 30 mg per day. Various cases of adulteration of DS and SF for weight loss and sport nutrition have been revealed: undeclared addition of synephrine in the form of bitter orange extract, addition of synthetic synephrine, its isomers or analogs. The main method for the determination of synephrine and other biogenic amines in DS and SF is high performance liquid chromatography with ultraviolet and/or mass detection. Conclusion. The data presented in the review confirm the feasibility of developing an official method for determination of main protoalkaloids and monitoring of DS and SF for the content of synephrine and other citrus protoalkaloids on its basis.

pH-induced changes in activity and conformation of NADH oxidase from Thermus thermophilus.

Thermus thermophilus NADH oxidase (NOX) activity exhibits a bell-shaped pH-dependency with the maximal rate at pH 5.2 and marked inhibition at lower pH. The first pH transition, from pH 7.2 to pH 5.2, results in more than a 2-fold activity increase with protonation of a group with pKa=6.1+/-0.1. The difference in fluorescence of the free and enzyme-bound flavin strongly indicates that the increase in enzyme activity in a pH-dependent manner is related to a protein-cofactor interaction. Only one amino acid residue, His75, has an intrinsic pKa approximately 6.0 and is localized in proximity (<10 A) to N5-N10 of the isoalloxazine ring and, therefore, is able to participate in such an interaction. Solvent acidification leads to the second pH transition from pH 5.2 to 2.0 that results in complete inhibition of the enzyme with protonation of a group with an apparent pKa=4.0+/-0.1. Inactivation of NOX activity at low pH is not caused by large conformational changes in the quaternary structure as judged by intrinsic viscosity and sedimentation velocity experiments. NOX exists as a dimer even as an apoprotein at acidic conditions. There is a strong coupling between the fluorescence of the enzyme-bound flavin and the intrinsic tryptophans, as demonstrated by energy transfer between Trp47 and the isoalloxazine ring of flavin adenine dinucleotide (FAD). The pH-induced changes in intrinsic tryptophan and FAD fluorescence indicate that inhibition of the FAD-binding enzyme at low pH is related to dissociation of the flavin cofactor, due to protonation of its adenine moiety.

Detergent-solubilized bovine cytochrome c oxidase: dimerization depends on the amphiphilic environment.

The extent to which bovine cytochrome c oxidase (COX) dimerizes in nondenaturing detergent environments was assessed by sedimentation velocity and equilibrium. In contrast to generally accepted opinion, the COX dimer is difficult to maintain and is the major oligomeric form only when COX is solubilized with a low concentration of dodecylmaltoside, i.e., approximately 1 mg/mg protein. The dimer form is intrinsically unstable and dissociates into monomers with increased detergent concentration, i.e., >5 mg/mg protein. The structure of the solubilizing detergent, however, greatly alters detergent effectiveness by inducing either monomerization or aggregation. Triton X-100 is most effective at solubilizing COX, but it destabilizes COX dimers, even at low concentration. Undecylmaltoside, decylmaltoside, and octaethyleneglycolmonododecyl ether (C(12)E(8)) are less effective at solubilizing COX. Each prevents COX aggregation at high detergent concentration, but also destabilizes the COX dimer. Other detergents, e.g., Tween 20, sodium cholate, sodium deoxycholate, CHAPS, or CHAPSO, are completely ineffective COX solubilizers and do not prevent aggregation even at 10-40 mg/mL. The transition from dimers to monomers depends on many factors other than detergent structure and concentration, e.g., protein concentration, phospholipid content and pH. We conclude that the intrinsic dimeric structure of COX can be maintained only after solubilization with low concentrations of dodecylmaltoside at near neutral pH, and even then precautions must be taken to prevent its dissociation into monomers.

Detergent-solubilized Escherichia coli cytochrome bo3 ubiquinol oxidase: a monomeric, not a dimeric complex.

The protein molecular weight, M(r), and hydrodynamic radius, R(s), of Triton X-100-solubilized Escherichia coli cytochrome bo3 were evaluated by computer fitting of sedimentation velocity data with finite element solutions to the Lamm equation. Detergent-solubilized cytochrome bo3 sediments as a homogeneous species with an S20,w of 6.75 s and a D20,w of 3.71 x 10(-7) cm2/s, corresponding to a R(s) of 5.8 nm and a M(r) of 144,000 +/- 3500. The protein molecular weight agrees very well with the value of 143,929 calculated from the four known subunit sequences and the value of 143,025 measured by MALDI mass spectrometry for the histidine-tagged enzyme. We conclude that detergent-solubilized E. coli ubiquinol oxidase is a monomeric complex of the four known subunits.

Detergent-solubilized monomeric and dimeric cytochrome bc1 isolated from bovine heart.

Mitochondrial cytochrome bc1 complex, isolated from frozen bovine heart, was solubilized with five different "non-denaturing" detergents: dodecyl maltoside, octaethylene glycol monododecyl ether (C12E8), Triton X-100, Tween 20, and sodium cholate. The hydrodynamic properties of the solubilized complex III's were then investigated by sedimentation analysis. Complex III exists as a stable and monodisperse dimer when it is solubilized in low ionic strength buffer with a low concentration of any of the five detergents. At pH 7.8, 20 degrees C, the protein sediments as a homogeneous species with an s(obs) of about 14 S. The protein molecular weight of the 14S particle, after correction for bound detergent, is 465,000 +/- 30,000 as measured by sedimentation equilibrium analysis. The aggregation state and/or homogeneity of cytochrome bc1 is strongly dependent upon the concentration of the solubilizing detergent and ionic strength. The enzyme becomes a homogeneous, monomeric complex with a protein molecular weight of 235,000 +/- 20,000 and an s(obs) of 10-10.5 S after it is solubilized in high concentrations of Tween 20 (more than 5 mg/mg of protein) and sodium chloride (more than 0.5 M). However, a heterogeneous mixture of subcomplexes is produced upon solubilization of the complex with high concentrations of the other detergents and 0.5 M NaCl. Monomerization of cytochrome bc1 by Tween 20 and 0.5 M NaCl has no effect on either the spectral properties, the subunit composition, or the enzymatic activity and is reversible since the dimeric 14S particle is regenerated upon removal of the high concentration of salt.(ABSTRACT TRUNCATED AT 250 WORDS)

Subunit analysis of bovine cytochrome bc1 by reverse-phase HPLC and determination of the subunit molecular masses by electrospray ionization mass spectrometry.

A sensitive and simple scheme was developed for the rapid separation of mitochondrial complex III subunits by reverse-phase high-performance liquid chromatography (reverse-phase HPLC). Ten of the 11 subunits of cytochrome bc1 complex were separated with nearly baseline resolution between each peak. Cytochrome b was precipitated by acetonitrile on the column and could not be analyzed; the 10 other polypeptides were positively identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrospray ionization mass spectrometry (ESI/MS). The ESI/MS-determined molecular masses for subunits II, VI, VIII, IX, and XI are in excellent agreement with previously reported values; i.e., all are within +/- 2 mass units per 10 kDa. None of the other subunits gave molecular masses that agree with the published sequence values. The molecular mass of subunit I is 49 236 Da, which is far greater than the molecular mass of 35,833 Da calculated from the reported DNA sequence [Gencic et al. (1991) Eur. J. Biochem. 199, 122-131]. The Fe-S protein (subunit V) gives two masses which differ by 60 mass units, presumably due to either the partial loss of the two sulfur atoms or microheterogeneity. Neither mass agrees with the sequence value, the larger mass being 39 mass units lower than expected from the sequence. The molecular masses of subunits VII and X are 81 and 129 Da larger, respectively, than those calculated from their sequences [Borchart et al. (1986) FEBS Lett. 200, 81-86; Schägger et al. (1983) Hoppe-Seyler's Z. Physiol. Chem. 364, 307-311].(ABSTRACT TRUNCATED AT 250 WORDS)

A new spectral intermediate in cyanide binding with the oxidized cytochrome c oxidase.

Reaction of cyanide with oxidized cytochrome c oxidase at a low concentration of the ligand and pH > 8 reveals an initial phase, not reported earlier, associated with a small blue shift of the absorption spectrum, which is followed by a conventional red shift of the heme alpha(3+)3. The initial blue shift resembles the spectral changes induced under the same conditions by low concentrations of azide and it is not observed in the presence of 0.3 mM azide. It is suggested that, similarly to NO, cyanide and HN3 cannot only bind to heme alpha 3 but to Cu(2+)B as well, perturbing the spectrum of alpha(3+)3 indirectly. A rapid binding to Cu(2+)B could provide the long-sought intermediate in the cyanide reaction with heme alpha(3+)3, the existence of which is implied by the Michaelis-Menten type kinetics of the latter process.

Some aspects of fluorescence and microcalorimetric studies of cytochrome C oxidase.

Effects of the solubilizing detergent type, pH and temperature on the structure of cytochrome c oxidase have been studied by the intrinsic fluorescence and scanning microcalorimetry methods. The data obtained allow to conclude that the enzyme solubilization by lauryl maltoside gives a more native preparation in comparison with that obtained by solubilization in Tween 80.

Spectral shifts of cytochrome c oxidase induced by complexons.

Ca2+-chelating agents, such as EDTA and ATP, are shown to bring about a rapid spectral response of the oxidized cytochrome c oxidase due to reversal of the Ca2+-induced red shift of the gamma- and alpha-absorption bands of the ferric enzyme. In addition, complexons are found to bring about Ca2+-independent, slow irreversible spectral changes indicative of a conformational transition of cytochrome oxidase. 1 mol EDTA per mol enzyme is sufficient to produce the maximal effect even in the presence of excess Ca2+, indicating high specificity of interaction. It is suggested that the conformation of cytochrome c oxidase may be regulated by the tightly bound "non-redox' metal ions (Mg, Zn, Cux) known to be present in the enzyme. These ions might be involved in specific binding of physiological effectors with chelating properties, such as ATP.

[A slow spectral shift of cytochrome c oxidase induced by EDTA and o-phenanthroline]. / Medlennyi spektral'nyi sdvig tsitokhrom c-oksidazy, vyzyvaemyi EDTA i o-fenantrolinom.

Low concentrations of EDTA (in the presence of Ca2+ excess) or o-phenanthroline cause a blue shift of the oxidized cytochrome oxidase Soret absorption band. The effect develops within approximately 2 hours and does not depend on EDTA concentration provided the complexon is in a molar excess over the enzyme. It is suggested that the enzyme spectral characteristics depend on the presence of some tightly bound heavy metal ions which can stabilize one of the spectrally distinct conformations of cytochrome c oxidase.

Conformational change of cytochrome a3 induced by oxidized cytochrome c.

Cyanide binding with the oxidized resting Yonetani-type cytochrome c-oxidase followed spectrophotometrically reveals a relatively rapid initial phase the rate of which shows saturation behaviour with respect to [HCN] and secondary slower absorption changes to a first approximation independent of the ligand concentration. Oxidized cytochrome c greatly accelerates the initial phase of cyanide binding but does not affect significantly contribution or rate constant of the slow phase. The same effect is exerted by poly-L-lysine. Within a framework of a reaction mechanism assuming Cu2+B to be the initial HCN-binding site, cytochrome c3+ and other polycations are likely to bring about a conformational-change of cytochrome oxidase resulting in an increased affinity of Cu2+B for HCN. This could occur by virtue of loosening a bond between Cu2+B and one of its endogenous ligands facilitating displacement of the latter by HCN.

[Isolation and characteristics of peptide fractions from the rat brain in ischemia]. / Vydelenie i kharakteristika fraktsii peptidov iz mozga krys pri ishemii.

The low-molecular peptide fractions are obtained from the brain of experimental and control animals by the method which includes sedimentation of high-molecular compounds and gel-filtration on Sephadex G-10. Ischemia of the rat brain sharply changes the amino acidic composition of the isolated peptide fractions. An inhibitory effect of the studied fractions on the activity of DNA-dependent RNA polymerase as well as their effect on the heat denaturation of exogenous DNA are established.

[Protein synthesis and oxidative activity of the brain in oxygen deficiency]. / Sintez belka i okislitel'naia aktivnost' mozga v usloviiakh kislorodnoi nedostatochnosti.

Biochemical and electron microscopic research methods showed disorders of the metabolism and the ultrastructure of the rat brain under the impact of experimental hypoxia and anoxia. Two to 3.5 hours after the exposure, the synthesis of protein in the neuronal fractions was impaired and the activity of the oxidative enzymes in the subfractions of synaptosomes and mitochondria was inhibited. The tissue ultrastructure was also impaired which was most pronounced in the synaptic processes of neurones. The data indicate fine morphochemical correlations at the subcellular level in the period immediately following post-hypoxic brain survival.