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[170] Pteridine content of bacteria including photosynthetic bacteria
[170]
PTERIDINE CONTENT OF BACTERIA
599
[ 170] P t e r i d i n e C o n t e n t of B a c t e r i a I n c l u d i n g Photosynthetic Bacteria
By H. S. FORREST Principle The 2-amino-4-hydroxypteridinyl radical is resistant to oxidation by alkaline permanganate. Most naturally occurring pteridines contain this radical in more-or-less modified form. Compounds with substituents at the 6-position (e.g., biopterin, neopterin) yield the 6-carboxylic acid on oxidation; compounds in which the pyrazine ring of the pteridine moiety is reduced, are oxidized to the stable aromatic form; compounds with substituents on the pyrazine nitrogens probably lose these by oxidation and decarboxylation. Thus the procedure converts all pteridines in the cell or culture fluid into a few derivatives that are easily separable from each other and other fluorescent permanganate oxidation products; since most naturally occurring pteridines have carbon substituents at the 6-position, 2-amino-4-hydroxy-6-carboxylic acid is the major oxidation product. Synthetic material is readily available for comparison, quantitatively and qualitatively.
Method Cultures (using 1 liter of culture fluid) at appropriate stages are harvested by centrifugation, and the cells are washed three times with distilled water. The combined supernatant solution, and/or the cells are again suspended in distilled water, made alkaline with KOH, and heated on a steam bath; a saturated solution of KMn04 is added, about 10 ml at a time. Oxidation is complete when the solution remains red for about 3 hours. The volume of KMn04 solution needed for complete oxidation may vary from 10 to 150 ml, and the time required for oxidation, from 2 to 20 hours. Excess KMn04 is destroyed by adding ethanol or methanol, and MnO~ is removed by filtration and washed with ethanol-concentrated ammonia (2:1). The washings are evaporated to remove ethanol and ammonia, and the concentrated solution is combined with the original filtrate. The whole is brought to pH 3 with acetic acid, and the solution is treated with sufficient charcoal (Darco G60) to absorb all fluorescent materials. The charcoal is collected by centrifugation (at which time it is convenient to check the supernatant solution for fluorescence using a long wavelength UV light) and washed three times with 0.1 N acetic acid. If the amount of charcoal is small, it may be suspended in water and applied directly as a streak to Whatman
600
PTERIDINES, ANALOGS, AND PTERIN COENZYMES
[171]
No. 3 MM filter paper. With large amounts, it is more convenient to elute the charcoal with ethanol-concentrated ammonia (2:1), concentrate the eluate, and apply it as a streak to the filter paper. The chromatogram is developed with n-propanol-1% ammonia (2:1) ; the resultant blue-fluorescent bands are cut out from the dried chromatogram, the fluorescent materials are eluted from them with 1% ammonia, and small samples of the eluates are rechromatogramed against authentic standards (2-amino4-hydroxypteridine and 2-amino-4-hydroxy-6-carboxypteridine) in three solvent systems: n-propanol-1% ammonia (2:1); n-butanol-acetic acidwater (4:1:5), and 4% aqueous ammonia. The amounts of pteridines obtained are then estimated on the original eluate by fluorescence in a standard fluorometer [appropriate filters, for example, are (Corning numbers): primary, 5860; secondary, 3389]. In most cases, the amounts obtained are not sufficient to allow estimation by ultraviolet absorption (using the characteristic 360-nm peak of such compounds in 0.1 N sodium hydroxide); but when this is possible, the results are in good agreement with those obtained by fluorescence.
[ 171 ] R a d i o i s o t o p e A s s a y f o r R e d u c e d P t e r i d i n e s : the Phenylalanine Hydroxylase Cofactor B y GORDON GUROFF
Reduced pteridines can serve as cofactors for a number of oxygenase reactions. 1 In two cases it has been shown that the natural cofactor is indeed a reduced pteridine. In rat liver the cofactor for phenylalanine hydroxylation is reduced 2-amino-4-hydroxy-6-(L-erythro-l,2-dihydroxypropyl)pteridine (L-erythrobiopterin).2 In Pseudomonas the same reaction is catalyzed by reduced 2-aminod-hydroxy-6-(L-threotrihydroxypropyl)pteridine (L-threobiopterin).3 Because of the wide distribution of pteridines in nature, it is reasonable to assume that most of the reactions for which pteridines can serve as cofactors will be found to be pteridine-dependent in their native state. In view of the increasing awareness of and interest in pteridine-requiring reactions, methods for the determination of pteridine levels in biological materials have become valuable. The following method takes advantage of the ability of reduced pteridines to serve as cofactors for the phenyl1S. Kaufman, Ann. Rev. Biochem. 36, 171 (1967). S. :Kaufman,Proc. Natl. Acad. Sci. U.S. 50, 1085 (1963). G. Guroff and C. A. Rhoads, J. Biol. Chem. 244, 142 (1969).
PTERIDINE CONTENT OF BACTERIA
599
[ 170] P t e r i d i n e C o n t e n t of B a c t e r i a I n c l u d i n g Photosynthetic Bacteria
By H. S. FORREST Principle The 2-amino-4-hydroxypteridinyl radical is resistant to oxidation by alkaline permanganate. Most naturally occurring pteridines contain this radical in more-or-less modified form. Compounds with substituents at the 6-position (e.g., biopterin, neopterin) yield the 6-carboxylic acid on oxidation; compounds in which the pyrazine ring of the pteridine moiety is reduced, are oxidized to the stable aromatic form; compounds with substituents on the pyrazine nitrogens probably lose these by oxidation and decarboxylation. Thus the procedure converts all pteridines in the cell or culture fluid into a few derivatives that are easily separable from each other and other fluorescent permanganate oxidation products; since most naturally occurring pteridines have carbon substituents at the 6-position, 2-amino-4-hydroxy-6-carboxylic acid is the major oxidation product. Synthetic material is readily available for comparison, quantitatively and qualitatively.
Method Cultures (using 1 liter of culture fluid) at appropriate stages are harvested by centrifugation, and the cells are washed three times with distilled water. The combined supernatant solution, and/or the cells are again suspended in distilled water, made alkaline with KOH, and heated on a steam bath; a saturated solution of KMn04 is added, about 10 ml at a time. Oxidation is complete when the solution remains red for about 3 hours. The volume of KMn04 solution needed for complete oxidation may vary from 10 to 150 ml, and the time required for oxidation, from 2 to 20 hours. Excess KMn04 is destroyed by adding ethanol or methanol, and MnO~ is removed by filtration and washed with ethanol-concentrated ammonia (2:1). The washings are evaporated to remove ethanol and ammonia, and the concentrated solution is combined with the original filtrate. The whole is brought to pH 3 with acetic acid, and the solution is treated with sufficient charcoal (Darco G60) to absorb all fluorescent materials. The charcoal is collected by centrifugation (at which time it is convenient to check the supernatant solution for fluorescence using a long wavelength UV light) and washed three times with 0.1 N acetic acid. If the amount of charcoal is small, it may be suspended in water and applied directly as a streak to Whatman
600
PTERIDINES, ANALOGS, AND PTERIN COENZYMES
[171]
No. 3 MM filter paper. With large amounts, it is more convenient to elute the charcoal with ethanol-concentrated ammonia (2:1), concentrate the eluate, and apply it as a streak to the filter paper. The chromatogram is developed with n-propanol-1% ammonia (2:1) ; the resultant blue-fluorescent bands are cut out from the dried chromatogram, the fluorescent materials are eluted from them with 1% ammonia, and small samples of the eluates are rechromatogramed against authentic standards (2-amino4-hydroxypteridine and 2-amino-4-hydroxy-6-carboxypteridine) in three solvent systems: n-propanol-1% ammonia (2:1); n-butanol-acetic acidwater (4:1:5), and 4% aqueous ammonia. The amounts of pteridines obtained are then estimated on the original eluate by fluorescence in a standard fluorometer [appropriate filters, for example, are (Corning numbers): primary, 5860; secondary, 3389]. In most cases, the amounts obtained are not sufficient to allow estimation by ultraviolet absorption (using the characteristic 360-nm peak of such compounds in 0.1 N sodium hydroxide); but when this is possible, the results are in good agreement with those obtained by fluorescence.
[ 171 ] R a d i o i s o t o p e A s s a y f o r R e d u c e d P t e r i d i n e s : the Phenylalanine Hydroxylase Cofactor B y GORDON GUROFF
Reduced pteridines can serve as cofactors for a number of oxygenase reactions. 1 In two cases it has been shown that the natural cofactor is indeed a reduced pteridine. In rat liver the cofactor for phenylalanine hydroxylation is reduced 2-amino-4-hydroxy-6-(L-erythro-l,2-dihydroxypropyl)pteridine (L-erythrobiopterin).2 In Pseudomonas the same reaction is catalyzed by reduced 2-aminod-hydroxy-6-(L-threotrihydroxypropyl)pteridine (L-threobiopterin).3 Because of the wide distribution of pteridines in nature, it is reasonable to assume that most of the reactions for which pteridines can serve as cofactors will be found to be pteridine-dependent in their native state. In view of the increasing awareness of and interest in pteridine-requiring reactions, methods for the determination of pteridine levels in biological materials have become valuable. The following method takes advantage of the ability of reduced pteridines to serve as cofactors for the phenyl1S. Kaufman, Ann. Rev. Biochem. 36, 171 (1967). S. :Kaufman,Proc. Natl. Acad. Sci. U.S. 50, 1085 (1963). G. Guroff and C. A. Rhoads, J. Biol. Chem. 244, 142 (1969).