Design, synthesis, and characterization of a photoactivatable flavocytochrome molecular maquette

We report the construction of a synthetic flavo-heme protein that incorporates two major physiological activities of flavoproteins: light activation of flavin analogous to DNA photolyase and rapid intramolecular electron transfer between the flavin and heme cofactors as in several oxidoreductases. The functional tetra-alpha-helix protein comprises two 62-aa helix-loop-helix subunits. Each subunit contains a single cysteine to which flavin (7-acetyl-10-methylisoalloxazine) is covalently attached and two histidines appropriately positioned for bis-his coordination of heme cofactors. Both flavins and hemes are situated within the hydrophobic core of the protein. Intramolecular electron transfer from flavosemiquinone generated by photoreduction from a sacrificial electron donor in solution was examined between protoporphyrin IX and 1-methyl-2-oxomesoheme XIII. Laser pulse-activated electron transfer from flavin to meso heme occurs on a 100-ns time scale, with a favorable free energy of approximately -100 meV. Electron transfer from flavin to the lower potential protoporphyrin IX, with an unfavorable free energy, can be induced after a lag phase under continuous light illumination. Thus, the supporting peptide matrix provides an excellent framework for the positioning of closely juxtaposed redox groups capable of facilitating intramolecular electron transfer and begins to clarify in a simplified and malleable system the natural engineering of flavoproteins.     

Analysis and monitoring oftrans-isomerization by IR attenuated total reflectance spectrophotometry

Attenuated total reflectance for IR determination oftrans-isomers in fats appears to have distinct advantages over procedures currently used. The AOCS standard method CD 14-61 requires weighing and quantitative dilution of a sample with carbon disulfide before spectrophotometric analysis at 10.3 μm. In contrast, according to the attenuated total reflectance analytical procedure, one neither weighs nor dilutes but merely fills the cell with oil and reads at 10.3 μm. In addition to analyses fortrans-isomers in liquid oils, margarines and shortenings, attenuated total reflectance enables one to monitortrans-development continuously during hydrogenation. The presence of catalyst in unfiltered hydrogenated oils does not interfere with attenuated total reflectance measurements in contrast to classical transmission measurements. Unfiltered oil from the hydrogenator can be circulated through the attenuated total reflectance cell to recordtrans-isomerization during the reaction. ARS, USDA.     

Composition of mixed octadecadienoates via ozonolysis,chromatography and computer solution of linear equations

An approach to the analysis of 55 possible nonconjugated positional isomers of octadecadienoic acid is described and tested with mixtures of individual synthetic methyl esters. In the first example, by ozonolysis a sevencomponent mixture consisting ofcis,cis 5,12-, 6,10-, 6,11-, 6,12-, 7,12-, 8,12-, and 9,12-octadecadienoates was converted to aldehydes, aldehyde-esters and dialdehydes. These fragments were separated on a 50 m×0.2 mm free fatty acid phase (FFAP) vitreous silica capillary column. Equations for an arbitrarily restricted 12×15 matrix of linear simultaneous equations and a computer solution of the matrix provided the composition of the initial methyl octadecadienoate mixture. The power and significance of this method became apparent with the observation that only two of the seven isomers in the known mixture were resolved as single peaks by state-of-the-art capillary gas chromatography, but all seven were identified and estimated with acceptable error by the ozonolysis-capillary gas chromatography-computer procedure. In a generalized approach to the analysis of the 55 possible nonconjugated isomers, a computer program selects the appropriate matrix of linear simultaneous equations based on the aldehyde data supplied by the analyst. Twenty of 21 combinations of seven isomeric esters taken five at a time have been analyzed to assess the efficiency of the method. To illustrate applicability at this stage of development, the method has been used to analyze the diene products of the hydrazine reduction of γ-linolenic acid and the diene products from the biological desaturation of isomeric monoenes. The possibility of distinguishing geometric and positional isomers of 18∶2 has been opened by the observed separation ofcis-andtrans-unsaturated aldehydes and aldehyde-esters. Presented in part at the 16th World Congress of the International Society for Fat Research, October 4–7, 1983, in Budapest, Hungary, and at the American Oil Chemists' Society Meeting, May 15, 1986, in Honolulu, Hawaii.     

Selective hydrogenation of soybean oil: IV. Fatty acid isomers formed with copper catalysts

Two samples of soybean oil hydrogenated with copper-containing catalysts at 170 and 200 C were analyzed for their natural and isomeric fatty acids. Methyl esters of the hydrogenated oils were separated into saturates, monoenes, dienes and trienes by countercurrent distribution between acetonitrile and pentane-hexane. Monoenes were further separated intocis- andtrans-isomers on a silver-saturated resin column. Double bond location in these fractions was determined by a microozonolysis-pyrolysis technique. The diene fraction was separated with an argentation countercurrent distribution method, and linoleate was identified by infrared, ozonolysis and alkaliisomerization data. The double bonds in thecis-monoenes were located in the 9-position almost exclusively. However, the double bonds in thetrans-monoene were quite scattered with 10- and 11-isomers predominating. About 86% to 92% of the dienes consisted of linoleate as measured by alkali isomerization. Other isomers identified as minor components includecis,trans andtrans, trans conjugated dienes and dienes whose double bonds are separated by more than one methylene group. No. Utiliz. Res. Dev. Div., ARS, USDA.     

Relative reduction rates of fatty acid isomers by hydrazine

Mixtures of fatty acids and of hydrocarbons have been reduced by hydrazine in ethyl alcohol solution at 50 C. Samples were removed during reduction for analysis and the relative reduction rates calculated by digital computer.cis-15-Octadecenoic acid is reduced 1.3 times as fast as itscis-9-isomer. No differences were found amongcis-6,9- and -12-octadecenoic acids.cis-3-Octadecene was also reduced 1.3 times as fast as thecis-9-isomer andcis-1-octadecene 8.6 times as fast. Elaidic acid was reduced 1.27 times as fast as oleic. Presented at the AOCS Meeting, Chicago, October 1967. No. Utiliz. Res. Dev. Div., ARS, USDA.     

Edible oil evaluation by room odor tests: A preliminary report

Room odors developed on heating edible fats in open vessels were evaluated and characterized by a 20 member odor panel. Edible fats tested were: special soybean salad and cooking oils, hydrogenated soybean oil and some commercial salad and cooking oils. Factors were investigated that affect reliability and reproducibility of the test and the acuity of the panel members. The effects of fry temperature and size of sample were investigated. The method has been applied to a study of hydrogenated and unhydrogenated soybean oil samples. Presented at the AOCS Meeting, Chicago, September 1970. No. Market. Nutr. Res. Div., ARS, USDA.     

Fluorescence as a measure of brown substances in soybean lecithin

Summary The fluorescence of soybean lecithin increases in a linear fashion with an increase in brown substances. Since the measurement of fluorescence is simple and rapid, this measurement should prove a useful index of browning. The term “lecithin” is used throughout this paper to refer to the mixtures of phosphatides, oil, etc., known in the trade as soybean lecithin. It does not refer to phosphatidylcholine. Presented at fall meeting of American Oil Chemists' Society, October 11–14, 1954, in Mineapolis, Minn. One of the branches of the Agricultural Research Service, U. S. Department of Agriculture.     

Preparation of 9,15-octadecadienoate isomers1

Linolenic acid was reduced with hydrazine to produce a mixture containing a max of dienoic acids. After methylation this mixture was separated into trienoic, dienoic, monoenoic, and saturated esters by countercurrent distribution (CCD) with acetonitrile and hexane. The dienoic ester was further fractionated by CCD with methanolic silver nitrate and hexane to separate purecis,cis-9,15-octadecadienoate and the equimixture ofcis,cis-9,12- and 12,15-octadecadienoates. Following isomerization of thecis,cis-9,15-octadecadienoate with selenium, the geometric isomers were fractionated by CCD with methanolic silver nitrate and hexane. Puretrans,trans and purecis,cis isomers were isolated, as well as an unresolved mixture ofcis,trans andtrans,cis isomers. The characteristics of these isomers and related compounds are compared as determined by CCD, IR absorption, and capillary gas-liquid chromatography (GLC). Presented at the AOCS meeting in Minneapolis, 1963. A laboratory of the No. Utiliz. Res. and Dev. Div., ARS, USDA.     

Selective hydrogenation of soybean oil with sodium borohydride-reduced catalysts

The reaction of metallic salts in aqueous solution with sodium borohydride produces finely divided metals that are catalytically active for hydrogenation. Salts of nickel, cobalt, palladium and platinum give active catalysts for the selective hydrogenation of soybean oil. Iron and silver salts, when reduced with sodium borohydride, show no activity at 200C and atmospheric hydrogen pressure. The cobalt catalyst produces the least amount of stearate. Incorporation of palladium, platinum, copper or chromium up to 2% enhance the activity of the nickel catalyst. Copper and chromium salts, when reduced together, form catalysts that hydrogenate linolenyl groups in soybean oil seven times more rapidly than linoleyl groups. No stearate formation is observed with these binary catalysts. Presented at the AOCS Meeting, Houston, April 1965. No. Utiliz. Res. Dev. Div., ARS, USDA.