Purification and properties of riboflavin-binding protein from the egg white of the duck (Anas platyrhynckos).

Riboflavin-binding protein was purified from the egg white of domestic duck and some of its properties were investigated. The protein was homogeneous by the criteria of gel filtration on Sephadex G-100 and electrophoresis on sodium dodecyl sulphate-polyacrylamide gels, had molecular weight of 36 000 ± 1000 and, unlike the chicken egg white protein (Mr 32 000 ± 2000), was devoid of covalently-bound carbohydrate. It was similar to the chicken riboflavin-binding protein in its behavior on ion-exchange celluloses and affinity to interact with the flavin and its coenzymes, but differed significantly in amino acid composition in that it completely lacked proline and contained less of methionine and arginine. The protein partially cross-reacted with the specific antiserum to chicken riboflavin-binding protein with a spur during immunodiffusion analysis.

Origin of the activation barrier in the process of hydrogen abstraction: the perturbation treatment

The perturbation treatment previously given is extended to explain the process of hydrogen abstraction from the various hydrogen donor molecules by the triplet nπ* state of ketones or the ground state of the alkyl or alkoxy radical. The results suggest that, as the ionization energy of the donor bonds is decreased, the reaction is accelerated and it is not influenced by the bond strength of the donor bonds. The activation barrier in such reactions arises from a weakening of the charge resonance term as the ionization energy of the donor bond increases.

Purification and physicochemical, kinetic and immunological properties of allosteric serine hydroxymethyltransferase from monkey liver.

The homogeneous serine hydroxymethyltransferase purified from monkey liver, by the use of Blue Sepharose affinity chromatography, exhibited positive homotropic co-operative interactions (h = 2.5) with tetrahydrofolate and heterotropic interactions with L-serine and nicotinamide nucleotides. The enzyme had an unusually high temperature optimum of 60 degrees C and was protected against thermal inactivation by L-serine. The allosteric effects were abolished when the monkey liver enzyme was purified by using a heat-denaturation step in the presence of L-serine, a procedure adopted by earlier workers for the purification of this enzyme from mammalian and bacterial sources. The enzyme activity was inhibited completely by N5-methyltetrahydrofolate, N5-formyltetrahydrofolate, dichloromethotrexate, aminopterin and D-cycloserine, whereas methotrexate and dihydrofolate were partial inhibitors. The insoluble monkey liver enzyme-antibody complex was catalytically active and failed to show positive homotropic co-operative interactions with tetrahydrofolate (h = 1) and heterotropic interactions with NAD+. The enzyme showed a higher heat-stability in a complex with its antibody than as the free enzyme. These results highlight the pitfalls in using a heat-denaturation step in the purification of allosteric enzymes.

NMR studies of phase transition in ammonium hydrogen bischloroacetate, NH4H(CH2ClCOOO)2

Proton second moment (M2) and spin-lattice relaxation time (T1) of Ammonium Hydrogen Bischloroacetate (ABCA) have been measured in the range 77-350 K. A value of 6.5 G2 has been observed for the second moment at room temperature, which is typical of NH4+ reorientation and also a second moment transition in the range 170-145 K indicates the freezing of NH4+ motion. The NMR signal disappears dicontinuously at 128 K. Proton spin-lattice relaxation time (T1) Vs temperature, yielded only one sharp miniumum of 1.9 msec which is again typical of NH4+ reorientation. A slope change at 250 K is also observed, prbably due to CH2 motion. Further, the FID signal disappears at 128 K. Thus the Tc appears to be 128 K (of two reported values 120 K and 128 K). Activation energies have been calculated and the mechanism of the phase transition is discussed.

Hydrogen- 1 and Carbon- 13 Magnetic Resonance Studies of Nonactin-Calcium Complex

For an understanding of the cation selectivity and general binding characteristics of macrotetralide antibiotic nonactin (NA) with ions of different sizes and charges, the nature of binding of divalent cation, Ca2+, to NA and conformation of the NA-Ca2+ complex have been studied by use of 270-MHz proton nuclear magnetic resonance ('H NMR) and carbon-13 nuclear magnetic resonance (13C NMR). The calcium ion induced significantly large changes in chemical shifts for H7, H2, H3, and H5 protons of NA and relatively small changes for H18 and H2' protons. Changes in I3C chemical shift were quite large for carbonyl carbon, C,; it is noteworthy that in the NA-K+ complex, H2 and H2' protons practically do not show any change during complexation and carbonyl carbon shows a much smaller chemical shift change.

A comparative study of the photochemical hydrogen-abstraction reactions of a ketone and a thione

A perturbation treatment was developed for the comparative study of the abstraction of hydrogen from methane by formaldehyde and thioformaldehyde in their nπ* and ππ* states. Both in-plane and perpendicularplane reactions are considered. Results are qualitative but reveal clearly the prominent distinguishing features of these two photochemical processes in agreement with the experimental observations.

A simple procedure for purification of N-carbamoylputrescine: Application to assays of putrescine transcarbamoylase and agmatine iminohydrolase activities

The observation that N-carbamoylputrescine is quantitatively excluded on O-(carboxymethyl)-cellulose columns with simultaneous retention of putrescine and agmatine has been utilized to develop a sensitive radiometric assay for putrescine transcarbamoylase and a colorimetric assay for agmatine iminohydrolase. A simple procedure for obtaining bulk amounts of pure synthetic N-carbamoylputrescine by separation from putrescine and dicarbamoylputrescine on Dowex 50 (Na+) resin is described.

Purification of Antibodies Specific to a Dinucleotide Using the Hapten Bound to Deae Cellulose as an Affinity Column

The dinucleotide dpTpA held electrostatically on DEAE cellulose was used as an affinity column for the purification of dpTpA specific antibodies. Chromatography of the y-globulin fraction from dpTpA specific antisera on this column resulted in the retention of dpTpA specific antibodies which were later eluted along with the bound dpTpA using 1M NaC1. Dextran coated charcoal was the method of choice for the dissociation and removal of dpTpA bound to the antibodies. This method may extend itself to the purification of antibodies specific for other oligonucleotides.

Heat exposure and hypothyroid conditions decrease hydrogen peroxide generation in liver mitochondria

Exposure of rats to heat (39 +/- 1 degree C) decreased H2O2 generation in mitochondria of the liver, but not of the kidney or the heart. The effect was obtained with three substrates, succinate, glycerol 1-phosphate and choline, with a decrease to 50% in the first 2-3 days of exposure, and a further decrease on longer exposure. The dehydrogenase activity with only glycerol 1-phosphate decreased, which is indicative of the hypothyroid condition, whereas choline dehydrogenase activity remained unchanged and that of succinate dehydrogenase decreased on long exposure. The serum concentration of thyroxine decreased in heat-exposed rats. Thyroxine treatment of rats increased H2O2 generation. Hypothyroid conditions obtained by treatment with propylthiouracil or thyroidectomy caused a decrease in H2O2 generation and changes in dehydrogenase activities similar to those with heat exposure. Treatment of heat-exposed or thyroidectomized rats with thyroxine stimulated H2O2 generation by a mechanism apparently involving fresh protein synthesis. The results indicate that H2O2 generation in mitochondria of heat-exposed animals is determined by thyroid status.

Purification and partial characterization of microsomal cytochrome b555 from the higher plant Catharanthus-Roseus

Microsomal b-type hemoprotein designated, cytochrome b555 of C-Roseus seedlings was solubilized using detergents and purified by a combination of ion exchange chromatography and gel filtration to a specific content of 18.5 nmol per mg of protein. The purified cytochrome b555 was homogeneous and estimated to have an apparent molecular weight of 16500 on SDS-PAGE. The absorption spectrum of the reduced form has major peaks at 424, 525 and 555 nm. The α-band of the reduced form is asymmetric with a pronounced shoulder at 559 nm. The spectrum of the pyridine ferrohemochrome shows absorption peaks at 557, 524 and 418 nm indicating that the cytochrome has protoheme prosthetic group. The purified cytochrome is autoxidizable and does not combine with carbon monoxide, azide or cyanide. It is reducible by NADH in the presence of NADH-cytochrome b555 reductase partially purified from C-Roseus microsomes.

Use of a volatile buffer at ambient temperature *1: Versatile approach to the purification of self-complementary synthetic deoxyoligonucleotides by reversed-phase high-performance liquid chromatography

Abstract is not available.

X-Pro peptides. A theoretical study of the hydrogen bonded conformations of (alpha-aminoisobutyryl-L -prolyl)n sequences

Intramolecularly hydrogen bonded conformations of (Aib-Pro)n sequences have been analysed theoretically. Both 4-1 (C10 and 3-1 (C7 hydrogen bonded regular structures are shown to be stereochemically feasible. Conformational energies for the helical structures have been estimated using classical potential energy methods. Both C10 and C7 conformations have very similar energies. Pyrrolidine ring puckering has a pronounced effect on the energies, and only Cv-endo puckered Pro residues can be accommodated. The theoretical calculations using spectroscopic data suggest that the recently proposed novel 310 helical conformation for benzyloxycarbonyl(Aib-Pro)4-methyl ester is in solution, is indeed energetically and stereochemically favourable.

Generation of hydrogen peroxide on oxidation of NADH by hepatic plasma membranes

The oxidation of NADH by mouse liver plasma membranes was shown to be accompanied by the formation of H2O2. The rate of H2O2 formation was less than one-tenth the rate of oxygen uptake and much slower than the rate of reduction of artificial electron acceptors. The optimum pH for this reaction was 7.0 and theK m value for NADH was found to be 3×10–6 M. The H2O2-generating system of plasma membranes was inhibited by quinacrine and azide, thus distinguishing it from similar activities in endoplasmic reticulum and mitochondria. Both NADH and NADPH served as substrates for plasma membrane H2O2 generation. Superoxide dismutase and adriamycin inhibited the reaction. Vanadate, known to stimulate the oxidation of NADH by plasma membranes, did not increase the formation of H2O2. In view of the growing evidence that H2O2 can be involved in metabolic control, the formation of H2O2 by a plasma membrane NAD(P)H oxidase system may be pertinent to control sites at the plasma membrane.

Hydrogen bonding with mono- and di-carbonyls

The electronic absorption and i.r. spectroscopic studies are reported for the hydrogen bonding systems involving alcohol and various ketones. It is shown that the hydrogen bonding abilities of ketones are determined by the extent of delocalization of the lone pair electrons in their non-bonding molecular orbitals. Evidence for the formation of very weak intermolecular hydrogen bonds between alcohol and the π-electron part of the dicarbonyls has also been presented from the i.r. studies in the 3400–3700 cm−1 region.