A common HPLC-PDA method for amino acid analysis in insects and plants.

A common method for analysis of 17 amino acids from various insect species and plant parts was standardized using HPLC-PDA. Prior to hydrolysis, lyophilization of test samples was found indispensible to remove excess moisture, which interferes in hydrolysis and separation of amino acids. After the hydrolysis of plant and insect samples, 500 and 100 µL of boiling HCl, respectively for reconstitution, and 20 µL of hydrolyzed samples used for derivatization, provided best results. Gradient profile of mobile phase and run time up to 65 min were standardized to (i) overcome the problems related to eluting underivatized sample part, (ii) optimize the use of mobile phase and run time, and (iii) get better separation of different amino acids. Analysis of Chilo partellus larvae reared on sorghum seedling powder based artificial diet indicated that arginine and histidine quantities were on par in both samples. However, methionine was higher, and leucine, isoleucine, lysine, phenylalanine, threonine and valine were lower in sorghum seedlings than in C. partellus larvae, suggesting compensation of these amino acids by the insect through voracious feeding, as is being expected from artificial diet. This method was found highly sensitive, reproducible and useful for the analysis of amino acids for better understanding of insect-plant interactions.

Extraction of bioactive principles from Mucuna pruriens seeds.

Mucuna pruriens (L.) DC. Syn. M. prurita Hook. (Papilionaceae) is used in male impotency, as aphrodisiac, in sexual debility, and as nervine tonic. It also possesses anti-parkinson property, possibly due to the presence of L-DOPA. In the present study, attempts were made to develop the suitable method(s) for extraction of L-DOPA/other active components from the seeds using different solvents. The various extracts were also screened for their neuroprotective and antioxidant activities. In addition, TLC and HPLC fingerprinting of the extracts for amino acid components were also developed for preliminary and sophisticated analysis. The L-DOPA could be obtained in good yield on extraction with EtOH-H2O mixture (1:1) using ascorbic acid as protector. Interestingly, n-propanol extract, which contained negligible amount of L-DOPA, had shown significant neuroprotective activity, suggesting that some components, other than L-DOPA, might also be responsible for anti-Parkinson property of seeds. The extract (MW-0100) containing mainly amino acids and water-ethanol extract (1:1) (MWEL-1299) showed promising antioxidant activity (EC50 = 2.5 microg) against DPPH radicals. MWEL-1299 also exhibited encouraging results against 1-methyl-4-phenylpyridinium ion (MPP+) toxicity. The TLC fingerprinting may be used to authenticate the plant material in herbal industry.

Chromium uptake by Saccharomyces cerevisiae and isolation of glucose tolerance factor from yeast biomass.

Fermentations with yeast Saccharomyces cerevisiae in semiaerobic and in static conditions with the addition of chromic chloride into the used molasses medium were analysed. It was proved that the addition of optimal amounts of CrCl3 into the basal medium enhanced the kinetics of alcohol fermentations. The addition of 200 mg/l CrCl3 into the medium stimulated both the yeast growth and the ethanol production in all experimental conditions. On the other hand, the results showed that Cr3+ ions were incorporated into yeast cells during fermentation. Under these conditions the accumulation of Cr3+ ions was performed by yeast cells during the exponential growth phase, and with enriched amounts of 30-45 microg/g(d.m) of cells. Yeast biomass enriched with chromium ions was extracted with 0.1 mol/l NH4OH assuming that the extracts had the glucose tolerance factor (GTF). Then the extracts were passed through a gel-filtration column in order to isolate and purify the GTF. The presence of GTF in the purified fractions was determined by measuring the absorbance at 260 nm. It is evident from the obtained results that the added purified fractions enhanced the rates of CO2 production as well as the glucose utilization during alcoholic fermentation. As expected, the enhancement of both rates depended on the amounts of extracts added to the fermentation substrate. Thus, it is evident that purified extracts contained the GTF compound, and that Cr3+ ions were bonded to the protein molecule.

Free sugars, mucilage and amino acids of leaves of hibiscus esculentum L. var. lady finger

Free sugars, mucilage and amino acids contents of leaves of Hibiscus esculentus L., var. Lady's finger [Adelmoschus esculentus Moench, okra] were studied. GLC and PC analysis of free sugars and mucilage hydrolysates revealed the presence of rhamnose, arabinose, xylose fructose, glucose, galactose, galacturonic acid and glucuronic acid. Moreover, analysis of free amino acids content and total amino acids of protein hydrolysate was carried out by HPLC technique. The major free amino acid is tyrosine [0.76%], while glutamic acid [2.22%], aspartic acid [1.76%] and arginine [1.21%] constitute the main amino acids in the protein hydrolysate of the leaves of H. esculentus L., var. Lady's finger

Aminoácidos näo protéicos de Ateleia glazioviana Baillon / Non-proteinogenic aminoacids from Ateleia glazioviana Baillon

Ateleia glazioviana Baillon, Leguminosae-Papilionoideae, árvore de ocorrência na regiäo de Palmeira das Missöes (RS), possui atividade tóxica para o gado, ictiotóxica e repelente de insetos, referida por populares do interior do estado. Do extrato de sementes e pericarpo alado, foram isolados três aminoácidos näo protéicos, dois dos quais identificados como ácido 1-aminociclobutano -1,3-dicarboxílico e Ð-acetilomitina

Electroforesis de aminoácidos a alto y bajo voltaje en agarosa / Electrophoresis of aminoacid in agarose at high and low voltage

Se ha desarrollado una nueva técnica para la separación electroforética de una solución de aminoácidos, compuesta por: taurina, ácido glutámico, serina, glicina, histidina, lisina y ß-alanina; que utiliza agarosa C como medio soporte y buffer fórmico-acético (pH=2,2). Las separaciones se realizan en bajo y alto voltaje (100-150 V y 350-800 V, respectivamente) obteniéndose en este último caso una mejor resolución, el tiempo empleado varía de acuerdo con las condiciones esperimentales y no supera los treinta minutos. El método proporciona información equivalente a la obtenida por la electroforesis tradicional, en papel a alto voltaje, con un menor costo, instrumental sencillo y, además, permite la densitometría directa de las separaciones coloreadas con nihidrina; se lo aplica a la separación de aminoácidos urinarios

Chemical composition of a mixture of single-cell protein obtained from Kluyveromyces fragilis and whey proteins

De la fermentación de suero de leche entera se obtuvo un producto consistente en una mezcla de biomasa de Kluyveromyces fragilis y proteínas coaguladas del suero. El producto tuvo una composición similar a la de productos lavados de que se informa en la literatura, con un alto contenido de proteína cruda y un bajo contenido de cenizas. Asimismo, acusó tambiém un alto contenido de aminoácidos azufrados y de triptofano, los que usualmente son limitantes en al biomasa de levadura. El contenido de lisina fue inexplicablemente más bajo de lo esperado resusltando ser el aminoácido limitante. La calificación química de la protéina fue 91%. Del producto de biomasa com proteínas de suero se obtuvo un concentrado proteínico con un rendimento de 80%. El contenido de proteína del aislado fue de 75% y el contenido de ácidos nucleícos se redujó en 90.8%. Los restos de pared celular también se redujeron considerablemente

Caracterización de la estructura primaria de la interleucina-2 humana recombinante / Characterization of the primary structure of the recombinant human interleukin-2

La interleucina-2 humana recombinante clonada y expresada en E. coli, y purificada mediante cromatografía líquida de alta eficacia (HPLC), fue analizada para verificar su estructura primaria. El análisis de aminoácidos de la proteína pura coincide con la composición teórica esperada. La proteína fue hidrolizada con bromuro de cianógeno, los péptidos separados por HPLC fueron secuenciados automáticamente y determinada su composición aminoacídica, confirmándose la estructura desde el extremo N-terminal hasta el residuo 56. Con el propósito de completar la secuencia de la proteína, esta fue reducida y carboximetilada. La proteína se sometió a digestión con endoproteinasa GLU-C (Staphyloccocus aureus V8) y una parte de esta digestión se incubó a continuación con tripsina. Los péptidos purificados mediante gel filtración (Biogel P-6) y HPLC en fase inversa, fueron secuenciados con la técnica manual de doble acoplamiento con dimetilaminoazobenzeno isotiocianato e isotiocianato de fenilo (DABITC/PITC). La purificación de la digestión tríptica de la proteína reducida y carboximetilada mediante fase inversa, suministró los péptidos para análisis en espectrometría de masas utilizando como fuente de ionización bombardeo con átomos rápidos (MS FAB). Tanto los resultados de la secuenciación como la espectrometría de masas confirman que la interleucina-2 obtenida en E. coli posee la estructura primaria reportada para esta proteína