A rapid and direct method for the quantitative determination of tryptophan in the intact protein
TL;DR: Tryptophan content of several pure intact proteins when treated with the above method gave values in good agreement with those reported by others.
Abstract: 1. A method is given for the quantitative determination of free tryptophan or tryptophan in the intact protein by treating with ninhydrin in a mixture of formic acid and hydrochloric acid (reagent b), for 10min at 100°C. Glycyltryptophan was used as a standard for the determination of tryptophan in the intact protein. The extinction at 390nm was linear in the range 0.05–0.5μmol for free tryptophan (∈7120) and 0.05–0.30μmol for glycyltryptophan (∈15400). 2. Free tryptophan in the presence of protein may be determined by treating with ninhydrin in a mixture of acetic acid and 0.6m-phosphoric acid (reagent a) for 10min at 100°C, the extinction being linear for tryptophan in the range 0.05–0.9μmol. N-Terminal tryptophan peptides also give the typical yellow product on treatment with reagent a. 3. Tryptophan content of several pure intact proteins when treated with the above method gave values in good agreement with those reported by others. A mean tryptophan content of 11.25 (s.e.m. ±0.08) μmol/100mg of protein was found in rat brain during development from 1 to 82 days after birth.
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TL;DR: This review attempts to integrate and correlate the widely scattered literature on ninhydrin reactions of a variety of structurally different compounds and provides a scientific basis for further improvements of this important analytical technique.
Abstract: The reaction of ninhydrin with primary amino groups to form the purple dye now called Ruhemann's purple (RP) was discovered by Siegfried Ruhemann in 1910. In addition, imines such as pipecolic acid and proline, the guanidino group of arginine, the amide groups of asparagine, the indole ring of tryptophan, the sulfhydryl group of cysteine, amino groups of cytosine and guanine, and cyanide ions also react with ninhydrin to form various chromophores of analytical interest. Since its discovery, extensive efforts have been made to apply manual and automated ninhydrin reactions as well as ninhydrin spray reagents to the detection, isolation, and analysis of numerous compounds of interest across a broad spectrum of disciplines. These include agricultural, biochemical, clinical, environmental, food, forensic, histochemical, microbiological, medical, nutritional, plant, and protein sciences. This reaction is unique among chromogenic reactions in that at pH 5.5 it results in the formation of the same soluble chromophore by all primary amines which react, be they amines, amino acids, peptides, proteins, and even ammonia. Because the chromophore is not chemically bound to the protein or other insoluble material, it is not lost when the insoluble substrate is removed by centrifugation or filtration after the reaction is completed. The visible color of the chromophore is distinctive and is generally not affected by the yellow colors present in many food, plant, and tissue extracts. Adaptations of the classical ninhydrin reaction to specialized needs in analytical chemistry and biochemistry include the use of acid, alkaline, and fluorogenic ninhydrin reagents. To cross-fertilize information among several disciplines wherein an interest in the ninhydrin reaction has developed, and to enhance its utility, this review attempts to integrate and correlate the widely scattered literature on ninhydrin reactions of a variety of structurally different compounds. Specifically covered are the following aspects: historical perspective, chemistry and mechanisms, applications, and research needs. A better understanding of these multifaceted ninhydrin reactions provide a scientific basis for further improvements of this important analytical technique.
504 citations
TL;DR: Study on the substrate specificity with ribonuclease as the substrate revealed that the staphylococcal protease specifically cleaves peptide bonds on the COOH-terminal side of either aspartic acid or glutamic acid.
478 citations
TL;DR: Highly purified alpha-actinin can be made by using the low ionic strength extraction procedure previously described and subjecting the crude alpha-Actinin fraction obtained with this extraction procedure to successive chromatography on DEAE-cellulose and hydroxyapatite and SDS-polyacrylamide gel electrophoresis.
200 citations
TL;DR: The polypedtide chains that comprise the subunits of the tonofilaments, or th alpha-keratin component, of bovine epidermis were fractionated by combination of chromatography on DEAE-cellulose and preparative polyacrylamide-gel electrophoresis on a molar basis.
Abstract: 1. The polypedtide chains that comprise the subunits of the tonofilaments, or th alpha-keratin component, of bovine epidermis were fractionated by combination of chromatography on DEAE-cellulose and preparative polyacrylamide-gel electrophoresis. 2. The seve polypeptide chains investigated had generalyy similar properties; all contained two residues per molecule of tryptophan and N-acetylserine was the common N-terminal amino acid residue. 3. On the basis of close similarities in alpha-helix content and amino acid composition, the polypeptide chains were classified into three distinct groups. Each group contained approximately one-third of the total polypeptides on a molar basis. The groups and designated polypeptides chain numbers were: group one, polypeptides 1a and 1b, which had moleculae weights of 58,000, contained about 25% alpha-helix, 86 glutamic acid and 8 cysteine residues per molecule, but which differed in net charge, extinction coefficients and tyrosine contents; group two, polypeptides 2, 3, and 4, which hadmolecular weights within thewithin the range of 52,00-56,000, contained about 48% alpha-helix, 54 glutamic acid and 6 cysteine residues per molecule, but which differed in extinction coefficients and tryosine contents; and group, polypeptides 5 and 6, which had molecular weights of 47000-48000, contained about 56% alpha-helix, 64 glutamic acid and 4 cysteine residues per molecule, but which differed in extinction coefficients and tyrosine contents...
134 citations
26 Sep 2018
TL;DR: This brief integrated overview surveys and interprets the current knowledge of the reported multiple analytical methods for free and protein-bound tryptophan in pure proteins, protein-containing foods, and in human fluids and tissues, the nutritional significance of l-tryptophan and its isomer d-tryptionan in fortified infant foods and corn tortillas as well the possible function of tryptophile in the diagnosis and mitigation of multiple human diseases.
Abstract: Tryptophan is an essential plant-derived amino acid that is needed for the in vivo biosynthesis of proteins. After consumption, it is metabolically transformed to bioactive metabolites, including serotonin, melatonin, kynurenine, and the vitamin niacin (nicotinamide). This brief integrated overview surveys and interprets our current knowledge of the reported multiple analytical methods for free and protein-bound tryptophan in pure proteins, protein-containing foods, and in human fluids and tissues, the nutritional significance of l-tryptophan and its isomer d-tryptophan in fortified infant foods and corn tortillas as well the possible function of tryptophan in the diagnosis and mitigation of multiple human diseases. Analytical methods include the use of acid ninhydrin, near-infrared reflectance spectroscopy, colorimetry, basic hydrolysis; acid hydrolysis of S-pyridylethylated proteins, and high-performance liquid and gas chromatography-mass spectrometry. Also covered are the nutritional values of tryptophan-fortified infant formulas and corn-based tortillas, safety of tryptophan for human consumption and the analysis of maize (corn), rice, and soybean plants that have been successfully genetically engineered to produce increasing tryptophan. Dietary tryptophan and its metabolites seem to have the potential to contribute to the therapy of autism, cardiovascular disease, cognitive function, chronic kidney disease, depression, inflammatory bowel disease, multiple sclerosis, sleep, social function, and microbial infections. Tryptophan can also facilitate the diagnosis of certain conditions such as human cataracts, colon neoplasms, renal cell carcinoma, and the prognosis of diabetic nephropathy. The described findings are not only of fundamental scientific interest but also have practical implications for agriculture, food processing, food safety, nutrition, and animal and human health. The collated information and suggested research need will hopefully facilitate and guide further studies needed to optimize the use of free and protein-bound tryptophan and metabolites to help improve animal and human nutrition and health.
129 citations