Destruction of Amino Acids during Filter Paper Chromatography

Brush, Miriam K.; Boutwell, R. K.; Barton, A. D.; Heidelberger, Charles

doi:10.1126/science.113.2923.4

Cited by 37 publications

(8 citation statements)

References 5 publications

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“…1 filter paper and chromatographed by the descending method for 18 hours, using phenol saturated with water as solvent and water saturated with phenol to saturate the atmosphere of the tank. The papers were then removed and dried in air at room temperature (5). The position of the amino acids was located by spraying the paper with a 0.1 per cent solution of ninhydrin in butanol and heating gently with an infra-red lamp.…”

Section: Methodsmentioning

confidence: 99%

Transaminase Activity in Human Blood

Karmen¹,

Wróblewski²,

LaDue³

1955

J. Clin. Invest.

1,609

387

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Enzymatic transamination consists of the enzyme catalyzed reversible transfer of the alpha amino nitrogen of an amino acid to an alpha-keto acid with the synthesis of a second amino acid and a second alpha-keto acid. Enzymes catalyzing different transamination reactions are found widely distributed in animal tissues and have been shown to change in activity in some tissues during disease (1-3). These observations prompted the present study to determine if transaminase activity could be demonstrated in human serum and blood cellular elements and, if so, to study any variations in activity of this enzyme in the blood of normal and diseased man. METHODS AND MATERIALSThe two transaminases found most active in animal tissues are:1. "Glutamic-oxalacetic transaminase" Aspartate + alpha-keto glutarate = glutamate + oxalacetate 2. "Glutamic-pyruvic transaminase"Alanine + alpha-keto glutarate = glutamate + pyruvate When aspartate or alanine are incubated with alphaketo glutarate and a source of enzyme, the rate of production of glutamate may be taken as a measure of transaminase activity. The amount of glutamate produced after a given incubation period under standardized conditions was measured by quantitative paper chromatographic analysis (4).One-tenth molar solutions of I-aspartate, I-alanine, and alpha-keto glutarate were prepared in 0.06 M phosphate buffer and the pH of the solutions adjusted to pH 7.6. For serum transaminase determinations, 0.5 ml. of clear, non-hemolyzed serum, 1.5 ml. of 0.06 M phosphate buffer, pH 7.6, and 0.5 ml. of either the alanine or aspartate solutions were incubated for ten minutes at 37°C. At this time, 0.5 ml. of the alpha-keto glutarate solution was added and the incubation continued for 18 hours. For whole blood hemolysate transaminase determinations, equal volumes of blood and distilled water were shaken together for ten minutes, 1.0 ml. of the hemolysate was added to 1.0 ml. of the phosphate buffer, and the substrates added as above. The time of incubation of the hemolysate substrate mixture was three hours.At the end of the incubation period, proteins were separated by adding 7.0 ml. of absolute ethyl alcohol, centrifuging for ten minutes, and washing the precipitate with 5 ml. of 70 per cent ethanol. The supernatant was evaporated to dryness over a water bath and the residue dissolved in 1.0 ml. of 0.06 M phosphate buffer. Aliquots of 0.05 ml. were then applied to Whatman No. 1 filter paper and chromatographed by the descending method for 18 hours, using phenol saturated with water as solvent and water saturated with phenol to saturate the atmosphere of the tank. The papers were then removed and dried in air at room temperature (5). The position of the amino acids was located by spraying the paper with a 0.1 per cent solution of ninhydrin in butanol and heating gently with an infra-red lamp.The areas of paper corresponding to glutamate were cut out, rolled, and placed in test tubes. Elution of the amino acid from the paper and quantitative color development with ninhydrin were perf...

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Section: Methodsmentioning

confidence: 99%

Transaminase Activity in Human Blood

Karmen¹,

Wróblewski²,

LaDue³

1955

J. Clin. Invest.

1,609

387

View full text Add to dashboard Cite

show abstract

“…of solution can be separated and identified on a 5inch-square sheet of filter paper irrigated in a No. 4 American Medical museum jar. A tert-butyl alcoholformic acid-water mixture is employed as the first solvent and a single phase phenol-ammonia-water mixture as the second solvent.…”

mentioning

confidence: 99%

Rapid Two-Dimensional Procedure

Rockland¹,

Underwood²

1954

Anal. Chem.

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“…Fowden and Penny (8) report that the recovery of certain amino acids was decreased by heat-drving of paper chromatograms. Brush et al (6) concluded from the study of radioautographs of alanine-2-C14 run in phenol that chromatograms run in this solvent should not be heated at all. Berry and Cain (2) conclude that the intensity of the ninhydrin color reaction does not decrease appreciably if the chromatograms are not heated over 85°C.…”

Section: Resultsmentioning

confidence: 99%