An accurate colorimetric method for measurement of sulfaminohexose in heparins and heparan sulfates

A modification of a method for hexosamine analysis is presented which adapts it to measurement of sulfaminohexose in heparins and heparan sulfates. Unlike methods of sulfaminohexose analysis based upon coupling with indole, the absorptivity of polymeric and monomeric hexosamines is identical. N-Sulfated hexosamines are specifically deaminated in 33% acetic acid to yield free 2,5-anhydromannose residues which are then coupled to the color reagent 3-methyl-2-benzothiazolinone hydrazone hydrochloride. The sulfaminohexose content of a variety of heparins and heparan sulfates was determined with this methodology and compared with the indole-coupling method. Interferences by amino acids, proteins, and neutral sugar were evaluated in the sulfaminohexose assay and in the originally reported procedure for total hexosamine analysis.     

The enzymic degradation of ovalbumin and its glycopeptides

1. Ovalbumin glycopeptides, freed from all amino acids other than aspartic acid and a small proportion of leucine by repeated digestion with Pronase, were hydrolysed by 1-aspartamido-beta-N-acetylglucosamine amidohydrolase (glycoaspartamidase) to the corresponding oligosaccharides. The glycoaspartamidase did not attack ovalbumin itself. 2. Ovalbumin, with mannose/hexosamine ratio 5:4, lost 1.5moles of N-acetylglucosamine and more than 2moles of mannose after incubation with alpha-mannosidase and beta-N-acetylglucosaminidase respectively. 3. In ovalbumin glycopeptides with approximate mannose/hexosamine ratios 5:3 and 5:4, one and two N-acetylglucosamine residues respectively were accessible to the action of beta-N-acetylglucosaminidase. 4. A mixture of alpha-mannosidase and beta-N-acetylglucosaminidase, acting on an ovalbumin glycopeptide with mannose/hexosamine ratio 5:3.7, removed nearly 4moles of mannose and 1.5moles of N-acetylglucosamine. 5. alpha-Mannosidase removed about 1.5moles of mannose from the ovalbumin oligosaccharide with mannose/hexosamine ratio approx. 5:3. The subsequent action of beta-N-acetylglucosaminidase liberated less than 1mole of N-acetylglucosamine and made at least 1mole further of mannose accessible to alpha-mannosidase action. 6. It is concluded that the carbohydrate moiety of ovalbumin is linked through a glycosyl group to asparagine. In a molecule with mannose/hexosamine ratio 5:4, there are two beta-N-acetylglucosamine residues linked together in a terminal position, followed by alpha-mannose. There is also present a side chain containing two alpha-mannose units.     

Hexosamine biosynthesis and accumulation by fungi in liquid and solid media

Hexosamine biosynthesis and incorporation into polysaccharides glyco-proteins have been studied in four species of fungi—Aspergillus niger, Penicillium citrinum, Cladosporium cladosporoides, and Fusarium moniliforme. Hydrolytic, recovery, and analytical methods are described for the estimation of hexosamine accumulation in fungal growth. Optimum yields of hexosamine from matrix are achieved by using hydrolysis with strong acid (8M hydrochloric acid) over a period of 2–3 h. For all fungal-growth studies, hexosamine was quantitated, after hydrolysis, by an automated, amino acid analyzer programmed for the separation of amino sugars. Methods were also developed, using gas-liquid chromatography (nitrogen-selective alkaline flame-ionization detector and trimethylsilyl derivatives of hexosamines), and the modified Morgan-Elson reaction of N-acetylated hexosamines. Both the amino acid analyzer and gas-chromatographic method quantify nmol amounts of hexosamine per mg of dry-weight sample. In all phases of the growth cycle, a linear correlation was found for the four fungi between the amount of hexosamine per unit time, age of culture, and quantity of mycelial biomass in liquid medium. With solid corn-meal as a fungal growth-medium, samples also demonstrated a linear correlation, but only between hexosamine biosynthesis and age of culture, as biomass was not determinable. Autolysis of hexosamine occurs to only a limited extent during late stationary-phase in liquid medium and on corn.     

Quantitative analysis of N-sulfated, N-acetylated, and unsubstituted glucosamine amino groups in heparin and related polysaccharides

A colorimetric procedure for quantitative determination of free and substituted glucosamine amino groups in heparin and related polysaccharides has been developed. The total content of hexosamine amino groups is determined by a modification of the method of Tsuji et al. (1969, Chem. Pharm. Bull. 17, 1505-1510); this method involves acid hydrolysis under conditions effecting complete removal of N-acetyl and N-sulfate groups, deaminative cleavage with nitrous acid, and colorimetric analysis of the resultant anhydromannose residues by reaction with 3-methyl-2-benzothiazolinone hydrazone (MBTH). N-sulfated glucosamine residues are cleaved selectively by treatment with nitrous acid at pH approximately 1.5 (J. E. Shively, and H.E. Conrad, 1976, Biochemistry 15, 3932-3942) and quantitated by the MBTH reaction. Under carefully controlled conditions, deamination at pH approximately 1.5 is highly specific for N-sulfated glucosamine residues, but an excess of reagent causes some cleavage of residues with unsubstituted amino groups as well. Deaminative cleavage at pH approximately 4.5 results in preferential degradation of unsubstituted glucosamine residues, but some cleavage (5-8%) of N-sulfated residues also occurs. However, analysis of the content of N-sulfated residues by the specific pH 1.5 procedure allows appropriate corrections to be made. From the value for total hexosamine content and the sum of N-sulfated and unsubstituted residues, the content of N-acetylated residues is calculated by difference. The modified deamination procedures, in combination with product analysis by the MBTH reaction, have been applied to several problems commonly encountered in the analysis and characterization of heparin.     

High-throughput analysis of hexosamine using a colorimetric method

A 96-well plate method was developed for analysis of total hexosamine content in biological samples. Four hexosamine monomer derivatives—glucosamine hydrochloride, glucosamine sulfate, galactosamine hydrochloride, and mannosamine hydrochloride—were examined for the linearity of their spectra in the concentration range specified in the assay. The hexosamine concentration analysis range was linear from 0.1 to 1 mM. The quantification of hexosamines from chitin and chitosan upon acid hydrolysis was also tested. Accurate quantification of glucosamine content in chitin and chitosan with different molecular sizes and degrees of acetylation was demonstrated using the new method.     

Isolation and characterization of pregnancy associated alpha2 glycoprotein]

A method using immunoadsorbents for the isolation of pregnancy-associated alpha2-glycoprotein (alpha2-PAG) from the extract of human placentae is described. The physical properties and the chemical composition of the purified protein are determined: alpha2PAG sediments with 11,5 S, has a molecular weight of 360 000 daltons and is composed of subunits having a molecular weight of 180000, which are held together by disulfide bonds. The isoelectric point was found to be pH 4,7 and the extinction coefficient (E1%1cm) was determined to be 9,7 at 277 nm. The carbohydrate content of the molecule amounts to 12,1% (hexose 6,0%, hexosamine 3,7%, fucose 0,06%, sialic acid 2,4%). An analysis of the amino acids is reported, too. The purified alpha2PAG was used to determine the absolute concentrations of this protein in a reference standard and in sera.     

The hypobranchial mucin of the whelk Buccinum undatum L. Properties of the mucin and of the glycoprotein component

1. The composition of the hypobranchial mucin from Buccinum undatum is reported. 2. The amino acid composition was determined; aspartic acid and glutamic acid contribute almost 24% of the total amino acids in the mucin. 3. Serine, threonine and alanine, in the proportions 2:1:1 respectively, were detected as N-terminal residues, implying the presence of at least four protein chains. 4. A glycoprotein component was isolated by phenol precipitation. 5. The glycoprotein contained 8% of neutral sugars comprising glucose, galactose, mannose and fucose, and 4.5% of hexosamine, comprising glucosamine and galactosamine in equal proportions. 6. A method is described for the preparation of glycopeptides from the glycoprotein. 7. The comparative biochemistry of the mucin is discussed.     

Glycoproteines sulfates des membranes de l'oeuf de poule et de l'oviducte Isolement et caracterisation de glycopeptides sulfatesSulfated glycoproteins form egg shell membranes and hen oviduct. Isolation and characterization of sulfated glycopeptides

Sulfated glycopeptides were isolated from pronaisc and tryptic digests of egg shell membranes and hen oviduct. They were precipitated by cationic detergents and separated by preparative electrophoresis, after removal of small quantities of glucuronoglycosaminoglycans detected only in the oviduct (isthmus and magnum). The principal isolated sulfated glycopeptides were divided according to increasing electrophoretic mobilities into two groups A and B. The homogeneity of the purified glycopeptides was confirmed by gel filtration and polyacrylamide gel electrophoresis.Glycopeptides from pool preparation of tissue are analysed and carbohydrate and amino acids average values are estimated. Hexosamines (mainly N-acetylglucosamine), hexoses (galactose, glucose, mannose) and fucose were found in Glycopeptides A. The molar ratio of hexose/hexosamine was 0.4. N-Acetylneuraminic acid and sulfate were also present in Glycopeptides A. The molar ratio of sulfate/hexosamine ranged from 0.1 to 0.25. The Glycopeptides A composition indicated the presence of chains with many glycosyl groups and a few of amino acids residues. The carbohydrate components of Glycopeptides B from egg shell membranes and magnum were found to be hexosamines (N-acetylgalactosamine and N-acetylglucosamine in equimolar proportions), hexoses (galactose mainly and glucose), N-acetylneuraminic acid, and fucose. The molar ratio of hexose/hexosamine was 1. Sulfate was also present and the molar ratio of N-acetylneuraminic acid and sulfate to hexosamine was ranged from 0.8 to 1. The main amino acid residues in these glycopeptides were serine and threonine with destruction of these hydroxyamino acids during alkali treatment. Glycopeptides B probably consist of short carbohydrate chains, linked to the polypeptide through O-glycosidic bonds involving N-acetylgalactosamine and serine and threonine. Approximately 40% of the amino acid residues were linked to carbohydrate chains.Glycopeptides B from egg shell membranes magnum and egg white were very similar in their carbohydrate and amino acid composition and in their properties.Gylcopeptides A from egg shell membranes, isthmus and magnum showed similarities and divergences especially in the amino acid composition. These results suggest that magnum and isthmus in oviduct are both concerned with the synthesis of egg shell membrane glycoproteins.     

A distinct terminal structure in newly synthesized chondroitin sulphate chains.

A method was developed for the analysis of non-reducing terminal structure of radiolabelled chondroitin sulphate chains with the aid of N-acetylgalactosamine 4-sulphatase ('terminal 4-sulphatase'), N-acetylgalactosamine 6-sulphatase ('terminal 6-sulphatase'), beta-glucuronidase and beta-N-acetylhexosaminidase. Studies with this method on the non-reducing terminal structure of [35S]sulphate- and [3H]glucose-labelled chondroitin sulphate chains from rat and chick-embryo cartilages showed that the presence of a high proportion of 4-sulphated hexosamine residues is a common feature of the termini of newly synthesized chondroitin sulphate chains. Of the non-reducing terminal 4-sulphated hexosamine residues, about 14% (chick embryo) or 46% (rat) contained an additional sulphate group at position 6. The internal portion of the chondroitin sulphate chains, in contrast, contained little or no 4,6-bis-sulphated hexosamine residue, suggesting that 4,6-bis-sulphated structure may play a role in biosynthetic control at the level of chain termination.     

The composition of the cyst wall of the beet cyst-nematode Heterodera schachtii

1. Cyst walls of the beet cyst-nematode (Heterodera schachtii Schmidt) were obtained by sieving a suspension of crushed cysts; about 15mg of dried cyst walls was obtained from 1000 cysts. 2. The cyst walls contained 68% protein calculated from nitrogen content. Glutamic acid, glycine, proline and hydroxyproline made up about 54% by weight of the amino acids obtained on acid hydrolysis. 3. Minor constituents of the cyst wall were hexosamine (3.3%), lipid (6%), carbohydrate (2%) and phenols (2%). The hexosamine was identified as galactosamine. 4. The cyst walls contained inorganic material (ash 17%), most of which was extractable with EDTA, but not with water. Major inorganic components were calcium and phosphorus (1.7% and 1.5% respectively, by weight). Carbon dioxide (about 1% by weight) was liberated from the cyst walls on acidification. 5. The cyst walls of H. schachtii and the potato cyst-nematode (Heterodera rostochiensis) contained different amounts of the same amino acids. They also differed in their inorganic content and in the nature of the hexosamine present.     

Production of heparin related glycosaminoglycans by an extablished mammalian cell line

A sulfated glycosaminoglycan has been isolated from the acid-soluble fraction of an established line of Chinese hamster fibroblasts grown in suspension culture. This material has a molecular weight between 5000 and 10,000, contains equimolar amounts of hexosamine and uronic acid (orcinol method), and about 0.6 sulfate groups per hexosamine residue. About 80% of the sulfate groups are N-sulfates on the basis of lability of the sulfate and the formation of equivalent numbers of free amino groups upon mild acid hydrolysis. The material is completely resistant to testicular hyaluronidase but is degraded to reducing monosaccharides and small oligosaccharides upon treatment with lyophilized cells of Flavobacterium heparinum that were grown on heparin. It is thought, therefore, to be related to the known N-sulfated glycosaminoglycans heparin and heparitin sulfate.     

Evidence that intracellular stages of Leishmania major utilize amino sugars as a major carbon source

Intracellular parasites, such as Leishmania spp, must acquire suitable carbon sources from the host cell in order to replicate. Here we present evidence that intracellular amastigote stages of Leishmania exploit amino sugars in the phagolysosome of mammalian macrophages as a source of carbon and energy. L. major parasites are capable of using N-acetylglucosamine and glucosamine as primarily carbon sources and contain key enzymes required for conversion of these sugars to fructose-6-phosphate. The last step in this pathway is catalyzed by glucosamine-6-phosphate deaminase (GND), which was targeted to glycosomes via a canonical C-terminal targeting signal when expressed as a GFP fusion protein. Mutant parasites lacking GND were unable to grow in medium containing amino sugars as sole carbohydrate source and rapidly lost viability, concomitant with the hyper-accumulation of hexosamine-phosphates. Expression of native GND, but not a cytosolic form of GND, in Δgnd parasites restored hexosamine-dependent growth, indicating that toxicity is due to depletion of glycosomal pools of ATP. Non-lethal increases in hexosamine phosphate levels in both Δgnd and wild type parasites was associated with a defect in promastigote metacyclogenesis, suggesting that hexosamine phosphate levels may influence parasite differentiation. Promastigote and amastigote stages of the Δgnd mutant were unable to replicate within macrophages and were either completely cleared or exhibited reduced lesion development in highly susceptible Balb/c mice. Our results suggest that hexosamines are a major class of sugars in the macrophage phagolysosome and that catabolism of scavenged amino sugars is required to sustain essential metabolic pathways and prevent hexosamine toxicity.     

Phosphonoglycoprotein from Metridium senile--heterogeneity of glycoproteins containing aminoethylphosphonic acid.

1. After separation by SDS gel-chromatography, analysis of AEP-containing glycoproteins from M. senile, indicated 66% amino acids with 220 AEP res./1000 res. and 30% carbohydrate for high mol. wt (greater than 10(7) forms and 80% amino acids with 25-50 AEP res./1000 res. and 10% carbohydrate for low mol. wt (2-4 x 10(4) forms. 2. Uronic acids, sulfate, lipid, and sialic acids were absent. 3. Mild base digestion released AEP-hexosamine containing oligosaccharides and destroyed ser-thr residues in the high mol. wt components. 4. Phosphonoglycoproteins appear to be acidic connective tissue components with AEP linked to hexosamine containing oligosaccharide side chains.     

Acid hydrolysis and quantitative determination of total hexosamines of an exopolysaccharide produced by <Emphasis Type="Italic">Citrobacter</Emphasis> sp.

During the hydrolysis of an exopolysaccharide (EPS) produced by Citrobacter sp., the maximum liberation of hexosamine was obtained with 6 m HCl at 115 °C in an autoclave for 1 h. The glycosidic bond energy and degree of acetylation of the hexosamine in EPS were approximately 77 kJ mol^–1 and 61%, respectively. Thermal destruction of the hexosamines and the effect of salt on the hexosamine determination could be minimized under the optimized hydrolytic conditions. Using a modified Elson–Morgan method, maximum total hexosamine concentration was determined to be 3.2 g l^–1 (29% of crude EPS) after 96 h of fed-batch culture.Revisions requested 18 August 2004; Revisions received 2 November 2004     

3H]myoinositol incorporation into phospholipids in liver microsomes from humans with and without type II diabetes. The lack of synthesis of glycosylphosphatidylinositol, precursor of the insulin mediator inositol phosphate glycan

A class of inositol phosphate-containing oligosaccharides (IPG) derived from a membrane glycan-phosphatidylinositol precursor (GPI) has been identified as a possible mediator of insulin action. Saltiel's laboratory has recently communicated an in vitro assay for the synthesis of GPI in rat liver microsomes. Herein we have established this method in rat and human liver microsomes, it being our end point to evaluate if the pool of GPI was normal in diabetes and if failure of insulin to generate IPG from GPI could be involved in the mechanism of insulin resistance in Type II diabetes. However, subsequent to the detailed study of [3H]myoinositol incorporation into phospholipids in liver microsomes from our study subjects, we demonstrated by gas chromatography/mass spectrometry analysis that the material reported to be GPI is a mixture of lysophospholipids that does not contain hexosamine, ethanolamine, or amino acids.     

New insights into the metabolic regulation of insulin action and insulin resistance: role of glucose and amino acids.

In primary cultured adipocytes, metabolic substrates such as glucose and amino acids have profound effects on modulating insulin's stimulatory actions on glucose uptake and protein synthesis. Insights into how substrates modulate insulin action were recently obtained when we discovered that the routing of incoming glucose through the hexosamine biosynthesis pathway leads to a refractory state over a period of several hours in which the ability of insulin to stimulate glucose uptake is severely impaired--a state known as insulin resistance. Glutamine:fructose-6-phosphate amidotransferase was found to play a central role in the development of insulin resistance as this enzyme catalyzes the first and rate-limiting step in the formation of hexosamine products. Collectively, these results are consistent with the idea that the hexosamine biosynthesis pathway serves as a glucose sensor coupled to a negative feedback system that can limit the extent of glucose uptake in response to hyperglycemic and hyperinsulinemic conditions.     

An antigen common to a wide range of bacteria. 2. A biochemical study of a "common antigen" from Pseudomonas aeruginosa

Common Antigen (CA) of Pseudomonas aeruginosa has been shown to be a protein composed of polypeptide subunits of a molecular weight (MW) of about 62 000. The MW of this protein was estimated to 665 000 by gel filtration on sepharose CL-6B, to 800 000 by electrophoresis on polyacrylamide gradient gels and to about 900 000 by ultracentrifugation, on a sucrose gradient. By analytical ultracentrifugation with Schlieren optics a sedimentation coefficient (S20 degrees, W) of 22.65 was calculated. The isoelectrical point was determined to pH 4.4. The antigen was decomposed on exposure to proteolytic enzymes. Polysaccharide, lipid, deoxyribonucleic acid or ribonucleic acid were not demonstrated in CA. The amino acid content of CA was determined, and no hexosamine or abnormal residues were observed. The amino acid content of CA was determined, and no hexosamine or abnormal residues were observed. The antigen was degraded when heated to 100 degrees C for 4 min or when exposed to pH below 4 or above 11 at 4 degree C. CA has been isolated from the cytoplasmic water-soluble fraction of disintegrated bacteria and only trace-amounts could be obtained from envelope fractions after solubilization with Triton X-100.     

Studies on the Cell Wall of Piricularia oryzae†

The chemical constituents of the cell wall of Piricularia oryzae, the pathogenic fungus of rice blast disease, were studied with the aids of chemical analysis, X-ray diffraction, infra-red absorption and enzymatic degradation. The sugar constituents were identified by chromatography as glucose (62%), mannose (4%), galactose (0.5%), and hexosamine (13%). The acidic amino acid rich protein was comprised 4.6% in the cell wall. The cell wall consists of at least three different polysaccharide complexes: a) α-Heteropolysaccharide protein complex containing mannose, glucose and galactose, b) β-1,3-Glucan containing β-1, 6-linked branch, c) Chitin like substance.     

Fluorometric quantitation of amino sugars in the picomole range.

A fluorometric method has been developed for the convenient and quantitative assay of amino sugars over the concentration range of 10 nm to 6 mm. Linear results are obtained for reaction mixtures containing 6 pmol to 60 nmol hexosamine. The procedure involves the condensation of amino sugars with the fluorogenic reagent o-phthalaldehyde, at alkaline pH in the presence of 2-mercaptoethanol. Relative fluorescence intensities are then determined using excitation and emission wavelengths of 340 and 455 nm, respectively. The presence of 2-mercaptoethanol in reaction mixtures not only enhanced sensitivity of the assay, but also defined the excitation/emission spectra. Under the conditions described, amino acids were also found to react with o-phthalaldehyde, yielding fluorescence intensities similar to those of amino sugars. These results suggest the applicability of fluorescence techniques in automated amino sugar analyses, as well as the potential interference of other compounds containing primary amines.     

Unique human glycoprotein, alpha1-microglycoprotein: isolation from the urine of a cancer patient and its characterization.

A human glycoprotein was isolated from the urine of a patient with plasma cell leukemia. It appears pure and homogeneous when examined by immunoelectrophoresis, sodium dodecyl sulfate (NaDodSO4)-polyacrylamide gel electrophoresis, gel filtration in 6 M guanidine hydrochloride (Gdn.HCl), and NH2-terminal amino acid sequence analysis. It has a brown color due to a tightly (most likely covalently) bound chromophore group(s) and migrates to the alpha1 region in immunoelectrophoresis. A molecular weight (mol wt) of 27 000 was obtained for the glycoprotein by gel filtration in 6 M Gdn.HCl. Its approximate mol wt determined by Na-DodSO4-polyacrylamide gel electrophoresis is 29 000 on 5% and 7.5% and 10% gels. Amino acid and hexosamine analyses showed that it is a glycoprotein and indicated that it contains four half-cystine residues per molecule. Based on the above observations we designated it "alpha1-microglycoprotein" (alpha1-MGP). Isoelectric focusing of alpha1-MGP showed a significant charge heterogeneity, although only a single NH2-terminal amino acid sequence was obtained for alpha1-MGP, i.e., Gly-Pro-Val-Pro-( )-Pro-Pro-Asx-Asx-Ile-Glx-Val-Glx-Glx-Asx-Phe-Phe-Ile-(Ser or Ala)-Arg. The alpha1-MGP was found in significant concentrations in the urine of many patients with neoplastic diseases.