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The determination of glucosamine by alkaline decomposition

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... Due to some difficulties caused by chitin insolubility in most solvents, it is usually detected indirectly as dGlcN or dGlcNAc (Ashwell, Brown, & Volk, 1965;Blix et al., 1948;Blumenthal & Roseman, 1957;Bosworth & Scott, 1994;Chen & Johnson, 1983;Dische & Borenfreund, 1950;Dous & Ziegenspeck, 1926;Dubois, Gilles, Hamilton, Rebers, & Smith, 1956;Elson & Morgan, 1933;Hicks & Newell, 1983;Hubbard, Seitz, & Mohr, 1979;Roseman & Daffner, 1956;Tracey, 1952;Tsuji, Kinoshita, & Hoshino, 1969 shift approximately 0.42 eV of the C 1s → π* acetamido (-NH(C--O)CH 3 ) group peak in holdfast chitin relative to amide (-NH-C(O)-) group position in collagen-like spongin. Furthermore, the spectra of the holdfast differ from those obtained for cellulose samples (reproduced with permission from Ehrlich, Rigby et al., 2013. ...
Article
Chitin is the second most abundant biopolymer and functions as the main structural component in a variety of living organisms. In nature, chitin rarely occurs in a pure form, but rather as nanoorganized chitin-proteins, chitin-pigments, or chitin-mineral composite biomaterials. Although chitin has a long history of scientific studies, it is still extensively investigated for practical applications in medicine, biotechnology, and biomimetics. The complexity of chitin has required the development of highly sensitive analytical methods for its identification. These methods are crucial for furthering disease diagnostics as well as advancing modern chitin-related technologies. Here we provide a summary of chitin identification by spectroscopic (NEXAFS, FTIR, Raman, NMR, colorimetry), chromatographic (TLC, GC, HPLC), electrophoretic (HPCE), and diffraction methods (XRD, WAXS, SAXS, HRTEM-SAED). Biochemical and immunochemical (ELISA, immunostaining) methods are described with respect to their medical application. This review outlines the history as well as the current progress in the analytical methods for chitin identification.
... at 100' (Stickland, 1951); dried bovine plasma albumin (N, 15.9%) from Armour and Co. Ltd., Hampden Park, Eastbourne, was used as standard. Ammonia was determined by nesslerization after distillation of the sample in a Markham still with alkaline phosphate + borate buffer (Tracey, 1952). Keto acids were estimated by the method of Friedmann tk Haugen (1943); individual keto acids were identified according to the paper chromatographic method of El Hawary & Thompson (1953). ...
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The survival characteristics of washed stationary phase Aerobacter aerogenes organisms suspended in buffered sodium chloride solution and stored at room temperature, or at 37° with aeration, depended on the medium used for growing the bacteria. Populations of bacteria harvested from tryptic meat broth or tryptone glucose medium remained almost completely viable for longer periods than bacteria from a simple ammonium salt + mannitol medium in which carbon was limiting. Analyses of washed freeze-dried preparations of freshly harvested bacteria showed that the amounts of protein, carbohydrate and ribonucleic acid present varied according to which of the above media was used for growth. During the initial stages of storage at 37°, when the viability of the population remained apparently unchanged, a progressive loss in bacterial dry weight occurred, due to degradation of these cell constituents. Endogenous glycogen was degraded and oxidized; bacteria which contained glycogen survived well. However, the addition of glucose to suspensions stored under aerobic or anaerobic conditions did not favour survival. Utilization of substances made available by degradation of various endogenous macromolecular constituents may be an important factor concerned with the survival of bacteria in unfavourable environments.
... The method for measuring hexosamine-N by steam distillation is based on the fact that glucosamine is rapidly decomposed in hot alkaline medium with the formation of ammonium (6). A method for estimating the hexosamine-N with an ammonia electrode without steam distillation was considered. ...
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The ammonia electrode method was used to characterize soil organic nitrogen. Amide-N, α-amino-N and hexosamine-N were estimated by measuring the ammonium-N derived from each organic-N with an ammonia electrode after hydrolysis of standard compounds followed by certain treatments. Each organic-N was recovered quantitatively from each standard compound in the presence and absence of soil. Twenty soil samples were analyzed for the above forms of organic nitrogen by both the distillation and electrode methods. The results obtained by the ammonia electrode method for each form of organic nitrogen agreed closely with those obtained by the distillation method. The electrode method was preferred for subsequent soil studies since it is simple and sensitive.
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1. Fraction I, a fraction containing acidic glycoproteins, isolated from guinea-pig serum, was digested with Pronase after removal of sialic acid and a major and a minor glycopeptide fraction were isolated by chromatography with Sephadex G-25 and G-50. 2. The major fraction was examined by various methods and shown to contain several glycopeptides. Estimates of molecular weight of the glycopeptide fractions were obtained. Although some variation appeared to occur, the glycopeptides were not grossly heterogeneous with respect to size. An average prosthetic group was estimated to contain about 15 sugar residues. 3. Aspartic acid was the principal amino acid present in the fractions and in all subfractions of the major fraction investigated. Where examined, ammonia was liberated on acid hydrolysis in approximately equimolar amounts to the aspartic acid present. The carbohydrate composition of the fractions was also determined. 4. The glycopeptides showed relatively little degradation in alkaline solution. 5. These results suggest that an N-acylglycosylamine bond involving aspartic acid forms the major type of linkage between carbohydrate and polypeptide. The isolation of a compound with the composition and chromatographic properties of 2-acetamido-1-(l-beta-aspartamido)-1,2-dideoxy-beta-d-glucose supports this view, and indicates that N-acetylglucosamine is the sugar involved in at least many linkages. 6. Fraction I contains some glycoproteins that are susceptible to Pronase and one or more others that resist digestion before the removal of sialic acid. A brief examination revealed some similarities between prosthetic groups derived from both kinds of glycoprotein.
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A yolk protein containing carbohydrate in eggs of the grasshopper, Locusta migratoria, was studied. The protein was homogeneous electrophoretically, soluble in acid and alkaline solution, and precipitated at neutral pH. The content of the protein was found to be about 77% of dry eggs. The carbohydrate moiety formed about 14% of the protein and seemed to be composed of 14 mannosyl and 2 glucosaminyl residues.A mixture of glycopeptides was prepared from the yolk protein by a procedure including extensive proteolytic digestion and deproteinization. The basic composition of these glycopeptides seemed to be 14 mannosyl, 2 glucosaminyl and 1 aspartyl residues.Several lines of evidence suggested that the polysaccharide was linked covalently to an asparaginyl residue of the protein moiety in the yolk protein.
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Neurospora sitophila 299 (pyridoxinbedrftig) zeigt mit steigendem Angebot an Pyridoxol HCl im Medium statistisch gesicherte Zunahmen an Protein, Zellwandsubstanzen (einschlielich Chitin) und Asche sowie Abnahmen an RNA und DNA. Der Lipidgehalt ist vom Pyridoxolangebot unabhngig. Der Durchmesser der Hyphen ist im Pyridoxinmangel statistisch signifikant grer als bei ausreichenden Gaben. Bei elektronenmikroskopischer Betrachtung gibt es keinen Anhalt fr Pyridoxin-induzierte Vernderungen der Zellwandstrke.With rising concentrations of pyridoxine in culture mediumNeurospora sitophila 299 (pyridoxineless) shows a significant increase of the relative amounts of protein, cell wall substances (incl. chitin), and ash, and a decrease of RNA and DNA. The lipid contents remain unchanged. The diameters of pyridoxine deficient hyphae are significantly greater than those of normal hyphae. Electron microscopic examination shows that B6-deficient cells do not contain a thinner or thicker than normal cell wall layer external to the membrane.
Chapter
This handbook is a reference guide for selecting and carrying out numerous methods of soil analysis. It is written in accordance with analytical standards and quality control approaches. It covers a large body of technical information including protocols, tables, formulae, spectrum models, chromatograms and additional analytical diagrams. The approaches are diverse, from the simplest tests to the most sophisticated determination methods in the physical chemistry of mineralogical and organic structures, available and total elements, soil exchange complex, pesticides and contaminants, trace elements and isotopes.As a basic reference, it will be particularly useful to scientists, engineers, technicians, professors and students, in the areas of soil science, agronomy, earth and environmental sciences. It is also relevant to those in related fields such as analytical chemistry, geology, hydrology, ecology, climatology, civil engineering and industrial activities associated with soil.
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1.1. Sodium chondroitin sulfate (SCS)-protein complexes of hyaline cartilage contain protein markedly different in amino acid composition from that of collagen.2.2. Light-scattering and viscosity data support the existence of a rodlike basic molecular unit. The molecular weight is 4.0 × 106, the length is 3700 A., the linear density is about 1000 avog./A. The structure proposed conceives the protein moiety as a core 3700 A. in length along which are distributed about 62 units of SCS, each of 50,000 molecular weight. Each linear SCS molecule, attached by one or more bonds to the protein core, retains its flexibility which allows an increase in average lateral chain extension with decrease in ionic strength.3.3. Aggregates, which have molecular weights as great as 50 × 106, are possibly formed by both lateral and end-to-end, hydrogen-bonded association of basic units. Additional protein may also be involved.4.4. The physiological significance of the results is discussed.
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A simple steam distillation method of estimating β-hydroxy-α-amino acids from the ammonium released by their reaction with alkaline periodate is described. It involves steam distillation with phosphate-borate buffer (pH 11.2) before and after treatments with periodic acid (to oxidize β-hydroxy-α-amino N to ammonium N) and with sodium arsenite (to reduce excess periodate), the ammonium liberated by the second distillation being determined by titration of the distillate with standard acid. The method is rapid and precise, and it gives quantitative results with serine, threonine, and other β-hydroxy-α-amino acids. When applied to protein hydrolyzates, it gives results in close agreement with those obtained by the periodate method of estimating β-hydroxy-α-amino acids proposed by Van Slyke, Hiller, and MacFadyen.
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Chemically intact cell walls of filamentous and yeast-like forms of Mucor rouxii were isolated. Comparative studies were made on their composition and structure to explore possible morphogenetic implications. Both types of cell walls exhibited a complex chemical composition consisting of polysaccharides (glucosamine, galactose, mannose and fucose), phosphate, proteins (at least 13 common amino acids), lipids (readily extracted and bound), purines and pyrimidines (RNA type), Mg2+ and Ca2+. Chitosan was the most abundant component of both types of cell walls. Chitin was present in smaller quantities. No qualitative differences were found between the two types of cell walls. Major quantitative differences were found in protein, purine-pyrimidine, and especially mannose content, all of which were higher in the yeast walls. Electron microscopy of ultrathin section of whole cells showed pronounced differences in thickness and fine structure of the walls. Whereas yeast walls were seemingly composed of two layers, no distinct layering was apparent in filamentous walls, which were only one tenth as thick as yeast walls.
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Full-text available
There is an urgent need for a rapid and reliable laboratory procedure capable of predicting soil N availability during land treatment of organic wastes or other sources of nitrogen. Soil systems which have been treated with organic wastes will likely to exhibit a relatively high potential to mineralize organic N into soluble forms under oxidation conditions. Large mineralizable and unsued N‐pools In soil systems may leach into the ground water or may be washed off with surface water, which in turn may lead to the degradation of body of water. Although several soll N availability indexes have been set forth, none of those tests have gained cohesive acceptance in the scientific community. Electro‐ultrafiltration (EUF) is one such procedure that has been proposed to effectively quantify soil PMN. This technique provides for extraction of NO3 and NH4 and of readily soluble N compounds from soils using the principles of Ultrafiltration and electrodialysis. The objective of this study was to assess the nature of the relationship between N extracted by the EUF technique and several other indexes of soil N availability for a group of benchmark soils from Nebraska. The data showed a correlation coefficient of 0.71 between the results obtained with the waterlogged method and those achieved using the EUF technique. However, the autoclave, KCI, pH 11.2 phosphate‐borate buffer, and NaHCO3‐UV methods were found more highly correlated (r ≥ 0.87) with the EUF technique than was the waterlogged method. The results obtained with the alkaline KMnO4 method yielded the lowest correlation coefficient (r= 0.25) with the EUF technique. The slopes of the regression equations between EUF and the chemical Indexes tested indicated that the EUF procedure was a relatively stronger extratant than the KCI method. Assessing soil N supplying capacity as a criterion represents a practical and efficient approach that can minimize the potential hazard of ground and surface water contamination for applying organic wastes or other N sources to the land.
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A simple method of determinations of three hexosamines is presented. The solutions of hexosamines heated in 1 n NaOH at 60°C for 60 min, absorb ultraviolet light with the maximum at 302 nm. A linear relationship exists between the concentration of hexosamine up to 100 μg and the absorbance at 302 nm. N-acetylated hexosamines, having been deacetylated, may be determined in this way. The characteristic absorption curve ranging from 225 to 360 nm with the maximum at 302 nm may be utilized as a qualitative criterion for identification of hexosamines and N-acetylated hexosamines after their deacetylation.
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material. The studywas initiated partlyinordertoinvestigate thenature oftheprotein-carbohydrate linkage, butthisgoal couldnot be achievedby themethodsthen available. Someprogress hasbeenmade inthis problemby more recentstudies and Johansen, Marshall & Neuberger (1960) havegiventhemost probable values forthemannose,glucosamine and acetyl contents ofthewholeprotein andofaglycopeptide isolated fromit.Theprobability thatthese aretheonlysugarspresentwasindicated. Itis thepurposeofthispapertodescribe thepreparationand some oftheproperties ofthisglycopeptide, andtoconsider thenatureofthechemical bondlinking thecarbohydrate to theprotein. A briefdescription ofthisworkwas reported earlier (Johansen, Marshall & Neuberger, 1958). Cunningham, Nuenke& Nuenke (1957)and Jevons(1958) havealsogivenshortaccountsof their findings onthesamesubject.
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This chapter discusses the aspects of the chemistry of the amino sugars. Several general methods have been employed for the synthesis of 2-amino-2-deoxy-aldoses; these respectively involve direct amination of suitable sugar derivatives, interconversion of sugar series, and ascent or descent of a series with or without concomitant amination or molecular rearrangements. Free amino sugars can be synthesized by the rearrangement of ketosylamines which carry no N-substituent. The rearrangement is catalyzed by weak organic acids, and takes place concurrently with a similarly acidcatalyzed, hydrolytic cleavage of the ketosylamine. Strong acids catalyze the hydrolysis and that weak acids (for example, benzoic and succinic acids) catalyze the rearrangement. Heyns synthesis of amino sugars is of particular theoretical interest since it is believed that the biosynthesis of amino sugars takes place in a related manner which involves the 6-phosphates. Considerable difficulty attends the isolation of the products, and over-all yields are often low.
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Some of the dialyzable products of the digestion of Micrococcus lysodeikticus cell walls by lysozyme and by a similar enzyme secreted by a Streptomyces have been isolated and their compositions determined. The two simplest substances released by both enzymes are (a) a di-saccharide of N-acetylmuramic acid-N-acetylglucosamine and (b) an N-acetylmuramic acid-N-acetylglucosamine complex. Seven peptide-acetyl-amino sugar complexes have been isolated. All seven compounds contain lysine, glutamic acid, glycine, alanine in the same molecular proportions as found in the original cell wall and a di-saccharide moiety of N-acetylmuramic acid and N-acetylglucosamine. One of the peptide-amino sugar complexes contains in addition a polysaccharide moiety [N-acetylmuramic acid-N-acetylglucosamine]10.
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THE usually accepted value for the amide nitrogen content of ovomucoid is 1.00 per cent1 or 21 residues per molecular weight of 28,800 gm. (ref. 2). This value was obtained by acid hydrolysis of the protein followed by distillation of the ammonia from mildly alkaline solution. It is known, however, that ovomucoid contains hexosamine and that the latter is deaminated to a greater or lesser extent by the methods used. It is thus likely that the reported values for the amide nitrogen content of ovomucoid, and those of other glycoproteins which have been determined by similar procedures, are too high.
Article
1. The isolation of two proteins from the seeds of kidney bean is described. 2. The individual steps in the purification procedure included: extraction of the seeds at pH9.0, dialysis, first against pH9.0 and then against pH5.0 buffers, high-voltage electrophoresis of the proteins soluble at pH5.0 and chromatography on Sephadex G-200, Sephadex G-75 and DEAE-Sephadex columns. 3. Of the two proteins isolated, the first and larger component was a glycoprotein and its carbohydrate part was mainly composed of d-mannose and d-glucosamine together with smaller amounts of arabinose, xylose and fucose. 4. The second protein component isolated was a trypsin inhibitor and was almost entirely devoid of sugars but contained a firmly bound pinkish-blue pigment. 5. The amino acid composition of the two proteins was determined. 6. The glycoprotein contained very little if any cyst(e)ine but was relatively rich in aromatic amino acids, whereas the trypsin inhibitor had an unusually high cystine content (nearly 15%) but was relatively poor in valine and in aromatic amino acids.
Article
The identity of the amino sugars in various soils has been studied by chromatographic techniques. Glucosamine and galactosamine were detected in every soil examined.
Chapter
IsolationMonosaccharide componentsMethylation techniqueReferencesDiscussion
Chapter
IntroductionHyrolysis of Amino Sugar Containing PolysaccharidesQualitative Determination of HexosaminesQuantitative Determination of Total HexosaminesQuantitative Determination of Glucosamine and Galactosamine in MixturesDetermination of N-Acetylhexosamines
Chapter
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INTRODUCCIÓN GENERAL El presente “Apéndice Analítico”1 se ha ideado como apoyo científico y técnico para profesionales como científicos, ingenieros, técnicos, profesores, estudiantes que, de una u otra forma, se interesan en el diagnóstico de la fertilidad del suelo y en la eficiencia de la fertilización. En el mismo, podrán encontrar unas discusiones críticas de los métodos de análisis de suelos más utilizados en estas materias, para tener mejores elementos de juicio que permitan una interpretación y aplicación racional y no dogmática de los resultados. Al desarrollar sus capacidades críticas, tanto el analista como el responsable de la interpretación de los resultados, tienen que conocer los alcances y los límites de la validez de estos análisis, para orientar mejor a los productores en la racionalización de la producción agrícola. En la consecución de este objetivo general se deben cumplir algunos objetivos específicos tales como: ‐ Seleccionar una serie de métodos de análisis accesibles a laboratorios modestos, dedicados principalmente al diagnóstico de la fertilidad de suelos cultivados y al seguimiento de los efectos del trabajo del suelo y de la fertilización. ‐ Poner a disposición del lector una bibliografía abundante que permita a los investigadores tener acceso a métodos especializados, que se hacen indispensables para su campo específico de investigación. En la primera parte de este “Apéndice Analítico”, se presentan los métodos físicos de fraccionamiento que incluye la granulometría y el fraccionamiento físico de la materia orgánica. El lector encontrará una discusión detallada de la ley de Stokes de la sedimentación, con sus limitantes y condiciones para su aplicación, así como los posibles errores que se pueden presentar. El fraccionamiento cuantitativo de los constituyentes orgánicos puede mejorar sensiblemente la calidad de los resultados analíticos y facilitar la interpretación de los procesos de mineralización, organización y humificación del carbono y del nitrógeno. En la segunda parte del Apéndice Analítico se presentan los métodos químicos, comenzando con la determinación de pH del suelo y la determinación de los requerimientos de cal. Seguidamente 1 El presente Apéndice Analítico procede en gran parte de la obra de M. Pansu y J. Gautheyrou*, quienes dieron su permiso para seleccionar y traducir elementos que pudieran ser de utilidad para completar esta obra de referencia sobre los suelos tropicales cultivados. Igualmente, fueron tomados algunos procedimientos del cuaderno de análisis rutinario de suelo usado por los laboratorios del INIA, elaborado por Julia Gilabert de Brito y Luis Nieves**, los cuales vienen a complementar la información presentada en la presente obra. Expresamos nuestro agradecimiento por su apoyo. * Marc Pansu et Jacques Gautheyrou, 2003 ‐ L’analyse du sol – minéralogique, organique et minérale. Springer, París, Berlín, Heidelberg, New York, 995 p. **Julia Gilabert de Brito y Luis Nieves 1990. Manual de Métodos Analíticos para caracterizar perfiles de suelos. CENIAP, Maracay, 101 p. 698 se describe el análisis elemental de C, H, N, tanto por vía húmeda como por vía seca o combustión. Este tipo de análisis es importante en la caracterización y cuantificación de la materia orgánica, elementos esenciales en el diagnóstico de la fertilidad potencial del suelo. Otro de los parámetros importantes en la caracterización de los suelos, es el intercambio iónico, por lo tanto, se presentan los métodos para la determinación de las capacidades de intercambio tanto catiónico como aniónico, y la cuantificación de los nutrientes en su estado llamado intercambiable al pH del suelo o a un pH estándar como el el pH 7 del buffer de acetato de amonio normal. Como es sabido, en el diagnóstico clásico de la fertilidad de suelos, la determinación de nutrientes se realiza por procedimientos de extracción utilizando diversas soluciones de reactivos más o menos agresivos, así como extractantes de diversa índole para cada nutriente o determinación. Este modelo de diagnóstico basado en procedimientos de extracción específicos afecta la productividad de los laboratorios y contribuye a aumentar los precios de los análisis. Ademas, estos procedimientos de extracción específicos de cada elemento o grupo de elemento presenta inconvenientes no solamente económicos sino también ecológicos, al aumentar indebidamente las cantidades de reactivos agresivos o tóxicos. En la tercera y última parte del Apéndice Analítico, se presenta una revisión bibliográfica sobre las determinaciones que se pueden hacer a partir de la llamada “solución de suelo”, compartimiento del sistema suelo‐planta que debería probablemente valorizarse más a futuro en el campo del diagnóstico de la fertilidad y de los efectos de la fertilización. Ello permitiría desarrollar procedimientos de extracción que usen menor cantidad de reactivos y que estos sean más amigables con el ambiente. Además, las mejoras en sensibilidad y exactitud de los métodos modernos ofrecen ahora límites de detección muy bajas para elementos solubles en el agua, con muestras de tamaño muy reducido, permitiendo en algunos casos detecciones secuenciales o simultáneas de bajas concentraciones de nutrientes. Actualizar los métodos tradicionales mejoraría la eficiencia de los análisis de suelo, disminuyendo el impacto ambiental de los reactivos evacuados en las aguas servidas de los laboratorios. Esto es especialmente importante para estudios de modelización de los flujos de nutrientes en el suelo, donde el seguimiento de la solución de suelo a lo largo de experimentos de larga duración requiere numerosos análisis químicos. Se acompaña la presentación de cada método de las consideraciones necesarias para un análisis crítico de los resultados. En efecto, es primordial conservar un punto de vista científico a lo largo de todo el proceso de diagnóstico de la fertilidad y los efectos de la fertilización. El proceso científico comienza en el momento de la toma de las muestras in situ y se termina por la lectura y la interpretación de los resultados. Los resultados deben luego interpretarse en función de un buen conocimiento del proceso analítico utilizado, porque sólo son un reflejo más o menos deformado de una realidad casi siempre compleja y cambiante. El científico reconstruye esta "realidad" según los modelos más o menos conscientes procedente de su formación científica inicial y de su experiencia profesional. Es necesario recordar estas exigencias, ya que los primeros químicos que pusieron a punto la mayoría de estos métodos de análisis de suelos, estaban, sobre todo, preocupados de obtener resultados fiables y reproductibles en el laboratorio, en una época en la que los límites de detección de los métodos analíticos eran mucho más elevados que ahora. Estos métodos clásicos reflejan en gran parte el nivel de conocimientos de un pasado lejano, donde se usaban métodos de análisis químicos poco sensibles.
Chapter
Carbohydrates are the most abundant and widely distributed food component. Carbohydrates include (1) monosaccharides (polyhydroxy aldehydes or ketones) among which are 5-carbon compounds, such as xylose or arabinose, and 6-carbon compounds, such as glucose and fructose, (2) oligosaccharides in which a hydroxyl group of one monosaccharide is condensed with the reducing group of another monosaccharide (if two sugar units are joined in this manner, a disaccharide results; a linear array of three to eight monosaccharides joined by glycosidic linkages gives oligosaccharides), and (3) polysaccharides that may be separated roughly into two broad groups, the so-called structural polysaccharides (i.e., cellulose, hemicellulose, lignin) that constitute or are part of rigid, mechanical structures in plants, and nutrient polysaccharides (i.e., starch, glycogen) that are metabolic reserves in plants and animals (Southgate 1991).
Chapter
The necessity for the inclusion of a section on this group of compounds was unfortunately realised late by the editors. As a consequence it proved impossible to secure a contribution from a recognised authority in the field. Since however it seems to the editors that it is a subject that has been unjustly neglected — particularly in Britain and the United States — it is hoped that this review will serve the purpose of directing interest towards this fascinating and almost certainly rewarding field of research.
Article
Chitin has long been defined as a substance that contains nitrogen, is insoluble in strong alkali solutions at 140° to 160°, and which on hydrolysis yields glucosamine and acetic acid. It has been established that some preparations of both animal and fungal origin are almost certainly straight chain, β 1–4 linked, polymers of acetylglucosamine (N-acetyl-2-amino-2-deoxy-d-glucose). On hydrolysis such a substance should yield 29,5% of its weight as acetic acid and 88% as glucosamine (106% as glucosamine hydrochloride). Its nitrogen content should be 6,9%. There is, as yet, no evidence that substances giving all the reactions of chitin described in Section B do not contain such a polymer, and X-ray evidence lends support to the conclusion that all so examined do contain it. A preparation that contains the theoretical amount of nitrogen and acetyl groups is a rarity owing to the difficulties of purification without degradation. It will be assumed in this article that materials satisfying the tests in Section B do in fact contain a pure acetylglucosamine polymer and that this is chitin. The reasons for this cautious approach will become apparent. It must be emphasised at the outset that to show that a material is “insoluble” and that on hydrolysis glucosamine is formed is no justification for assuming that it contains chitin. There are many polymers in both animals and lower plants that are insoluble under some conditions and that contain acetylglucosamine units. They are, however, mixed polymers and chitin is the only unmixed polymer of acetylglucosamine for which evidence has yet been secured.
Chapter
This chapter presents a procedure for the calorimetric analysis of sugars for the identification and determination of small quantities of carbohydrates obtained from biological materials. In general, such tests have been devised by heating aqueous solutions of the sugar with a strong acid, thereby converting it to furfural or a derivative of furfural. A color is then produced by the addition of an organic developer such as indole, orcinol, diphenylamine, or carbazole. Many of the reagents commonly employed for the determination of a specific class of sugars are suitable, with slight modifications, to measure total carbohydrates. Thus, orcinol, carbazole, indole, and diphenylamine have all been used for this purpose. Although these reactions logically require a separate classification, therefore they are discussed individually to prevent unnecessary duplication. For the determination of inulin diphenylamine reaction is employed, which is based on the Ihl-Pechmann reaction for fructose and is not to be confused with the diphenylamine test for DNA. In arsenomolybdate method of Nelson a colorimetric determination of the reaction product replaces the conventional iodometric titration of the reduced copper found in the Somogyi reagent, thus facilitating the handling of serial assays. In addition to the methods discussed in the chapter for the determination of pentoses, there are several alternate procedures which may be of value under special circumstances. The more important of these seem to be the methods of Roe and Rice and of Tracey employing p-bromoaniline and aniline, respectively, as the color-producing agent. These reactions are appreciably less affected by the presence of hexoses and uronic acids than is the case with orcinol.
Article
Numerous experiments (2, 7, 9–14,16) have been reported that drying a soil and rewetting it results in a flush of decomposition of soil organic matter and a flush of ammonium and then of nitrates. And if the soil is taken through a number of such cycles of drying, rewetting and incubation, the amount of nitrogen to be mineralized falls off slowly as the number of cycles through which it is carried increases. The magnitude of the flush is larger in lowland soils than in upland ones. Among lowland soils it is largest in the ill-drained paddy. In rough figures, abut 10 per cent of the organic nitrogen is decomposed through one cycle of drying and rewetting in the ever-flooded paddy in Japan. No completely satisfying reason can yet be given for this flush of decomposition, nor is it known what fraction of the organic matter is involved.
Article
The ammonia, hexosamine, and amino acid nitrogen fractions of soil hydrolyzates were determined with the Technicon autoanalyzer and the results compared with those from other more rapid methods of analysis. Ammonia determination by steam distillation agreed well with that by the autoanalyzer. Amino acid nitrogen, as determined by direct color development with ninhydrin and as ammonia after ninhydrin oxidation, agreed well with total amino acid nitrogen. Steam distillation was not a very satisfactory method of determining hexosamine nitrogen, probably because it was determined by difference between total distillable and distillable ammonia and the hexosamine nitrogen was small relative to the total nitrogen distilled.The soils tested contained approximately 20% acid-insoluble nitrogen. The acid hydrolyzate contained approximately 36% of total soil nitrogen as amino acid nitrogen, about 22% as ammonia nitrogen, and about 4% as hexosamine nitrogen. Approximately 20% of the soil nitrogen appeared in the hydrolyzate in a soluble form which was not identified by the methods used. A method is proposed which appears suitable for routine analysis of the nitrogen fractions of soils.
Article
The chemical nature and distribution patterns of the forms of N in a podzolic soil profile from central Sweden, developed under a stand of Norway spruce, were studied. Total N, native fixed NH4, acid hydrolysable-N, and the amounts of ammonia, hexosamine, and amino-acid N in the hydrolysate were determined. From 17 to 27 per cent of the N was insoluble in 6N HC1, the highest percentage being in the A2 horizon. Amino-acids in the acid hydrolysates decreased from 50 per cent of the total N content of the soil in the humus layer (A0) to 24 per cent in the B horizon. Amino-acid composition varied little in samples from different horizons. Hexosamine-N was 11–14 per cent of the total soil N, tending to increase with depth. Approximately 15 per cent of the total soil N was found in the soil hydrolysate as NH4. Values for native fixed NH4 extracted by N HF: N HCl were 34–80 ppm but were reduced to 10–17 ppm when corrected for the NH4 released by N HCl only. The figures thus obtained are considerably lower than those reported by other workers.
Article
A simple and sensitive method for determination of amide groups in proteins is presented. The method employs a combination of a simple microdiffusion technique and the indophenol method for ammonia determination. A similar ammonia analysis was used for total nitrogen determination after Kjeldahl digestion. The results from the amide group and total nitrogen analyses, together with previously published amino acid and carbohydrate analyses, were used to estimate a value of 12.4 for the specific extinction coefficient, E1%280 nm, of dopamine β-monooxygenase from bovine adrenal medulla.
Article
A SUBSTANCE reacting as a hexosamine but which is not glucosamine or galactosamine has been found in 6 N hydrochloric acid hydrolysates of a variety of bacterial cell walls by Cummins and Harris1 and in peptides isolated from spores of Bacillus megatherium, B. subtilis and B. cereus by Strange and Powell2. There is evidence that in all cases the same substance is involved, and recently in this laboratory a similar compound was isolated from acid hydrolysates of cells of Micrococcus lysodeikticus in sufficient quantity for comparison with that from spore peptide, which showed that it was identical in a number of properties. The amino-sugar would therefore seem to be widely distributed in bacteria. The base has now been obtained in crystalline form from acid hydrolysates of spore peptide (Fig. 1) by the use of ion-exchange resins. Investigation of the crystalline compound has shown that it is not a simple hexosamine ; for example, after treatment with ninhydrin according to the method of Stoffyn and Jeanloz3, followed by paper chromatography, a spot was obtained which reacted with aniline phthalate4 and other sugar spray reagents like a pentose but which had an RF value considerably lower than that of arabinose, lyxose, xylose or ribose. This product reduced ammoniacal silver nitrate but did not react with naphtho-resorcinol–trichloracetic acid5.
Article
Conventional steam-distillation techniques for fractionating the N in soil hydrolysates have generally indicated little variation in the chemical distribution or soil organic N, regardless or soil type, cropping, cultivation, or N-fertilization history. Nitrogen-15 tracer studies to evaluate these techniques showed that determinations of amino sugar-N are subject to serious underestimation, and that analyses for amino acid-N are vitiated by incomplete conversion or amino acid-N to NH4-N following incomplete removal of hydrolyzable NH4 and amino sugar-N. Diffusion methods were developed for fractionating the N in soil hydrolysates that are far more accurate and specific than steam distillation, while also being much simpler and more convenient. In these methods, total hydrolyzable N is measured by Kjeldahl digestion of the hydrolysate and diffusion of the digest with NaOH; diffusion is performed with MgO to determine hydrolyzable NH4-N; (NH4 + amino sugar)-N is recovered by diffusion with NaOH, after which amino acid-N is liberated by ninhydrin oxidation at pH <1.8 and recovered by diffusion with NaOH. Analytical accuracy and specificity were evaluated using a wide variety of purified organic-N compounds and by checking the recovery of N-15 added to soil hydrolysates as (NH4)(2)SO4, glucosamine, or glycine. Studies using a diverse set of soils showed that distillation and diffusion usually agreed to within 10% for quantitative analysis of total hydrolyzable N. NH4-N, and amino acid-N, whereas analyses of amino sugar-N were 74 to 317% greater by diffusion than by distillation.
Article
Chitin is a polysaccharide of β-(1→4)-linked 2-acetamido-2-deoxy-D-glucose (N-acetyl-D-glucosamine) that is found in the cell walls of fungi. In an effort to develop new methods to detect fungi in plant and animal tissues, chemical analyses based on fungal cell wall components have been evaluated. Chitin is not present in plant or most food animal tissues; therefore, the entire sample can be hydrolyzed and analyzed for fungal chitin. Acid, alkaline, and enzymatic hydrolysis have been used to cleave the β-(1→4)-glycosidic bond to produce glucosamine, chitosan, or N-acetylglucosamine. The major methods used to analyze these degradation products have included colorimetry; chromatography (gas chromatography, high performance liquid chromatography, amino acid analysis); microscopy, using fluorescent, nonfluorescent or immunofluorescent dyes; near-infrared spectroscopy; and titrametric assays. Chitin has been used to estimate and quantify fungal growth in plants, wood, grains, hay, and foods. There was an increase in the chitin content as the mold increased; however, the chitin assay showed more variability than other assays for detecting fungal contamination. The future use of the chitin assay will depend upon improvements in sensitivity, assay time, simplified methodology and equipment, and development of reliable conversion factors for converting chitin to fungal dry weight.
Article
A rapid steam distillation of assessing potentially available organic nitrogen in soil is described. It involves determination of the ammonia‐N produced by steam distillation of the soil sample with pH 11.2 phosphate‐borate buffer solution for 8 min. The method is simple and precise, and its results are not significantly affected by air‐drying or air‐dry storage of the soil sample before analysis. It is well suited for use in soil testing laboratories because it does not require extraction, filtration or transfer steps. Studies using 33 Brazilian soils showed that the results obtained by this method were highly correlated with those obtained by aerobic and anaerobic incubation methods of assessing potentially available organic nitrogen in soil.
Article
1. Nitrogen transformations during the decomposition of straw composted with ammonium carbonate have been studied by following the changes in ( a ) the amounts of inorganic and organic nitrogen; ( b ) the amounts of ammonia-, volatile base-, α-amino- and amino sugar-N liberated by acid hydrolysis of the organic nitrogen complexes; and ( c ) the amino acid composition of acid hydrolysates of the composts. 2. Synthesis of organic nitrogen during the biological decomposition of straw composted with ammonium carbonate is not accompanied by any gross change in the distribution of the forms of organic nitrogen. A large fraction of the organic nitrogen synthesized is in the form of protein; a smaller fraction is in the form of amino sugar.
Article
1. The amounts of amino sugar-N present in acid hydrolysates of six soils with nitrogen contents ranging from 0·17 to 2·82% have been estimated by colorimetric and alkaline decomposition methods. 2. Recovery of amino sugar-N after hydrolysis of chitin or glucosamine was found to be unaffected by the presence of soil during hydrolysis. 3. Substances known to interfere with the methods of amino sugar analysis employed were not detectable in the soil hydrolysates. 4. From the amounts of amino sugar-N liberated by acid hydrolysis it is deduced that 5·10% of the total-nitrogen of the soils examined was in the form of amino sugars. 5. The decomposition of amino sugars in soil has been studied by comparing the rates of decomposition of chitin, glucosamine, casein and yeast nucleic acid when incubated with soil under conditions found to produce rapid nitrification of ammonium sulphate. 6. Glucosamine and chitin are readily decomposed by soil micro-organisms but not so rapidly as casein or yeast nucleic acid.
Article
1. The chemical nature of the nitrogen in humic acid preparations isolated from 0·5M-sodium hydroxide and 0·1M-sodium pyrophosphate (pH 7·0) extracts of nine different soils has been studied by determining the amounts of acid-soluble-N, ammonia-N, amino-sugar-N and α-amino-N liberated by acid hydrolysis of the preparations and by paper chromatographic analysis of their acid hydrolysates. 2. Humic acid preparations isolated from alkali and pyrophosphate extracts of the same soil differ markedly in total nitrogen content and in nitrogen distribution after acid hydrolysis. The alkali-extracted preparations have a higher nitrogen content and a higher proportion of acid-soluble-N and α-amino-N. 3. A considerable fraction (20–60%) of the nitrogen in the preparations examined was not dissolved by acid hydrolysis. The major fraction of the nitrogen dissolved was in the form of amino-acids. 4. At least 31–48% of the nitrogen in the alkaliextracted preparations and 20–35% of the nitrogen in the pyrophosphate-extracted preparations was in the form of protein. From 3 to 10% of the nitrogen in the preparations was in the form of amino-sugars. 5. The results obtained by paper chromatographic analysis of acid hydrolysates of the preparations indicated that the protein materials in humic acids isolated from different soils by alkali or pyrophosphate are similar in their amino-acid composition. The following nineteen amino-acids were detected in every hydrolysate examined: phenylalanine, leucine, isoleucine, valine, alanine, glycine, threonine, serine, aspartic acid, glutamic acid, lysine, arginine, histidine, proline, hydroxyproline, α-amino-n-butyric acid, β-alanine, γ-aminobutyric acid and tyrosine. Two unidentified ninhydrin-reacting substances, oxidation products of cystine and methionine, and amino-sugars were also detected in every hydrolysate examined. A third unidentified ninhydrin-reacting substance and a substance provisionally identified as α,ε-diaminopimelic acid were found in some of the hydrolysates.
Article
Treatment of ovine-submaxillary-gland glycoprotein (OSM) with 0.1 N NaOH at 100° released about 89% of the carbohydrate groups (N-acetylneuraminyl (α,2 → 6)N-acetylgalactosamine; NANA ← GalNHAc) according to first-order reaction kinetics; k = 2.37·10−1 min−1 (half-time 2.9 min). The remaining prosthetic groups were liberated much more slowly; the first-order rate constant was 1.89·10−3 min−1. The carbohydrate-peptide linkages were also cleaved by NH2OH at pH 12.2 and 37° in a pseudo-monomolecular reaction, k = 2.3·10−3 min−1. The alkaline conditions on their own released the carbohydrate groups at a lower rate, k = 0.6·10−3 min−1. The disaccharides liberated were recovered quantitatively, mainly in form of their oxime which had protected them against destruction by alkali. Concomitant with the release of the disaccharides, hydroxamates were formed. Removal of the terminal negatively-charged N-acetylneuraminic acid (NANA) residues from OSM prior to NH2OH-treatment considerably increased the reaction rates: kNH2O− = 7·10−3 min−1, kOH− = 1.6·10−3 min−1. When about 89% of the carbohydrate groups had been liberated, the reaction was practically completed. OSM displayed no reducing power when tested with o-dinitrobenzene or Benedict's reagent under controlled conditions using GalNHAc as standard.The data presented provide further proof for the previous conclusion that the bulk of the disaccharide groups in OSM are joined through a glycosidic-ester linkage to the peptide. 11% of the groups are apparently linked in another fashion; the possibility of an O-glycosidic linkage to serine residues for this fraction is discussed. The amide-N of OSM is low, 2.6 μmoles/100 mg OSM. It was shown that crystalline NANA, as was previously found for bound NANA, liberates NH3 under the acid conditions used in the amide determination of proteins.
Article
DURING the course of investigations on the chemistry of the jelly coat of sea-urchin egg1,2, the analyses for hexosamine by paper partition chromatography3 were found to be negative in spite of the positive results obtained with the colorimetric method of Elson and Morgan4. This was found2 to depend on an interaction of the sugars with the amino-compounds, especially lysine, of the hydrolysate, resulting in a red colour with the same absorption maximum (at 530 mµ) as for glucosamine. The interaction of mixtures of sugars and certain amino-acids, especially lysine and glycine, on the hexosamine reaction has been pointed out before (for example, refs. 5 and 6); but in these cases, where the hexosamine values were rather high compared with the interfering substances, the discrepancies were small. In our case, however, the error apparently is unusually high. Since in other biological material similar conditions may exist, we thought this communication might be of some value.
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