Putrescine. analysis

The residue of crystalline putrescine dihydrochloride is rinsed onto a Buchner funnel with the aid of 100-200 ml. of absolute ethanol used in several portions. The last portions of ethanol are used as wash liquid for the crystals. The crystals are finally pressed dry and washed with 25 ml. of ether. The air-dried product weighs 53-55 g. (72-74%) and melts above 275. Analysis for chlorine indicates that the salt is anhydrous. Concentration of the filtrate to a volume of about 25 ml. yields an additional 1-2 g. total yield 73-77%. The entire synthesis may be completed in one day. 

Used to introduce chromophores into amines aids in resolution of putrescine, spermidine, and spermine by HPLC excess TsCI must be removed (by extraction with hexane, for example) before analysis Reference 41... 

The assay was carried out in phosphate buffer with radioactive putrescine, decarboxylated S-adenosylmethionine, and enzyme. Reactions were incubated at 37°C for 90 minutes and terminated by addition of perchloric acid. The solutions were clarified by centrifugation, and the polyamines were benzoyl-ated and extracted and then analyzed. Figure 9.55 shows the analysis of samples removed at zero time (blank) and in after 60 minutes incubation (sample) at 37°C. The appearance of radioactive spermidine is shown. The rate of product formation is shown in Figure 9.56. 

Nicotine biosynthesis also involves the incorporation of nicotinic acid (Fig. 2.2) (Robins et al., 1987), and the availability of this moiety can be as important in nicotine accumulation as that of the putrescine-derived portion. However, the enz)une responsible for the condensation of N-methylpyrrolinium with decarboxylated nicotinic acid, nicotine s)mthase (Friesen and Leete, 1990), was measured at only a very low level of activity, quite inadequate to account for the rates of nicotine accumulation observed in cultures. The molecular analysis of low-nicotine mutants of N. tabacum suggested the presence of regulatory genes (Me 1 and Me 2) governing the expression of nicotine bios)mthesis (Hibi et al, 1994). 

Teuber, M., Azemi, M.E., Namjoyan, R, Meier, A.-C., Wodak, A., Brandt, W. and Drager, B. (2007) Putrescine N-methyltransferases - a structure-function analysis. Plant Mol. Biol, 63, 787-801. 

Teuber M, Azemi ME, Namjoyan F, Meier A-C, Wodak A, Brandt W, Drager B. Putrescine N-methyltransferases—a structure-function analysis. Plant Mol. Biol. 2007 63 787-801. McLauchlan WR, McKee RA, Evans DM. The purification and immunocharacterization of N-methylputrescine oxidase from transformed root cultures of Nicotinia tabacum. Planta 1993 191 440-445. 

FIGURE 9.10. Evaluation of the peak purity of the putrescine-tryptamine system, (a) Experimental data set (b) Determination of the number of components by SVD (c) Study of impurities by window factor analysis (arrows indicate the time points at which spectra have been taken to be used as initial estimations and (d) Results of the resolution of the data set by MCR-ALS. 

Recently, a LC/ESI-MS method for analysis of tyramine, trypt-amine, 2-phenylethylamine, histamine, cadaverine, putrescine, spermidine, and spermine in wine without any sample pretreatment, was... 

Calystegins A3, Bi and B2 (257a-c) were isolated from Calystegia sepium. These alkaloids were also present in Convolvus arvenis and Atropa belladonna, and they were found to be catabolized by Rhizobium meliloti strain 41 [584], The structures of 257a-c were determined with HRMS, and H and NMR, and were confirmed by the synthesis and NMR analysis of model compounds [585], C-Putrescine served as a biosynthetic precursor for the calystegins [586],... 

The most commonly applied methods for the analysis of polyamines in erythrocytes make use of amino acid analyzers and HPLC techniques. A capillary gas chromatographic method with nitrogen-phosphorous detection was applied to the simultaneous determination of 1,3-diaminopropane, putrescine, cadaverine (Cad), spermidine (Sd), and spermine (Sp) in human erythrocytes. Blood samples, collected by venipuncture into EDTA containing Venoject tubes, were subjected to the removal of plasma by centrifugation and erythrocytes were washed three times with two volumes of 0.9% NaCl. The stability of polyamines in erythrocyte suspensions was also investigated. Quantification of polyamines was done by comparing the peak-area ratio of each analyte and its internal standard with that of the standard. The polyamine samples were eluted with 0.1 M hydrochloric acid solutions. The eluate was evaporated to dryness at 120°C under a stream of air and 200 each of acetonitrile and heptafluorobutyric anhydride were added. The isolation of derivatives... 

Bonilla, M., Enriquez, L. G., and McNair, H. M. (1995). Use of Cold On-Column Injection Technique for the Analysis of Putrescine and Cadaverine by Gas Chromatography. Pitteon 95. The Pittsburgh Conference. New Orleans, LA. 

An alternative for the same kind of analysis are silica columns that have been dynamically coated with a polyamine. Triethylene tetramine or natural amines such as spermidine or putrescine are suitable for this purpose. Small differences in selectivity are observed when different amines ate used. Dynamically coated packings give a more stable chromatography than do amino-propyl bonded phases, but the silica is still subject to slow dissolution. Therefore a silica with a strong skeleton ( = small specific pore volume) is recommended for this application. 

The choice of the column and the composition of the mobile phase were studied to obtain the best compromise between resolution and analysis time. The Nucleosyl CN reversed phase column was used because C 18 and C 8 retention times were excessive. The eluant composed of acetonitrile (40 %) and a buffer (60 %) (KH2PO4 0.05 M, EDTA 0.1 mM) gave the best separation of polyamines, when the pH was adjusted to 5.7. This pH is very important because changes in pH modify retention times of polyamines. Spermine and spermidine are the most affected by pH. In these conditions, the chromatograms showed satisfactory results and symmetrical peaks. The retention times were 40 minutes for spermine, 57 minutes for putrescine, 68 minutes for internal standard and 75 minutes for spermidine (Fig.l.). They were constant in repeated analyses. A good linear relationship (r = 0.99) existed between polyamine concentration and the peak height over the range 1 pmole to 10 nmole when the derivatization time was carefully controlled (5 min). 

---