The carbohydrate units of asialo-ovomucoid: structural features

The structural features of a heterogeneous glycopeptide fraction from asialo-ovomucoid have been investigated by methylation analysis of the fraction and of products obtained at each stage of its sequential degradation with exo-glycosidases. All glycopeptides in the fraction had a common core-structure beta-D-GlcpNAc-(1 leads to 4)-[beta-D-GlcpNAc-(1 leads to 2)]-alpha-D-Manp-(1 leads to 3)-[beta-D-GlcpNAc-(1 leads to 4)]-[beta-D-GlcpNAc-(1 leads to 2)-alpha-D-Manp-(1 leads to 6)]-beta-D-Manp-(1 leads to 4)-beta-D-GlcpNAc-(1 leads to 4)-beta-D-GlcpNAc leads to Asn. Heterogeneity in the fraction arose from variation in the amount of terminal galactose attached via a hexosaminyl residue to the alpha-D-Manp-(1 leads to 3) residue, and from limited variation in the number of terminal hexosaminyl groups attached to the alpha-D-Manp-(1 leads to 6) residue. One glycopeptide in the fraction contained the unusual feature of two different, triply-substituted mannosyl residues. Other structural features of the glycopeptide are discussed.     

Preparation and properties of a hemoglobin containing heme only in gamma subunits

A semihemoglobin containing prosthetic groups only in the γ-subunits (two hemes per tetramer) has been prepared by mixing together apo-α-subunits from hemoglobin A and native, heme-containing γ-subunits from hemoglobin F. The semihemoglobin has optical properties similar to those of hemoglobin F. The oxygen affinity of the semihemoglobin is lower than that of isolated γ-subunits but not as low as that of hemoglobin F, and the Hill coefficient of the semihemoglobin is near one. This semihemoglobin lends further support to the non-equivalence of the subunits in the hemoglobin tetramer.     

The acid-catalyzed dehydration of 1-benzylamino-1-deoxy-d-threo-pentulose, 1-dibenzylamino-1-deoxy-d-fructuronic acid,and d-glucuronic acid

Three compounds, 1-benzylamino-1-deoxy-d-threo-pentulose (1), 1-dibenzylamino-1-deoxy-d-fructuronic acid (2), and d-glucuronic acid (3) were converted into 2-furaldehyde in acidified, tritiated water. In the latter system, the 2-furaldehyde derived from 1 contained 13% of the activity of the solvent at the aldehyde carbon and 9% at positions 3–5 of the furan ring; that from 2 contained 8% at the aldehyde carbon and 29% at positions 3–5; and that from 3 contained 18% at positions 3–5 In deuterium oxide, the 2-furaldehyde derived from 1 contained 14 atom % of deuterium at position 3, 5% at position 4, and 0% at position 5. That from 2 contained 50% at position 3, 44% at position 4, and 7% at position 5. That from 3 contained 35% at position 3, 15% at position 4, and 5% at position 5. The data for 1 are discussed relative to prior data on incorporation collected for d-xylose Incorporation data for both 2 and 3 are qualitatively consistent with a decarboxylation step involving a β,γ-unsaturated, carboxylic acid intermediate. A mechanism for the decarboxylation of hexuronic acids is presented.     

The structure of the type-specific polysaccharide of Pneumococcus type XIX

The structure of the capsular polysaccharide of Type XIX Streptococcus pneumoniae (S-XIX) has been elucidated by ¹H- and ¹³C-n.m.r. spectroscopy. Mild hydrolysis of S-XIX with acid yielded a major oligosaccharide, the repeating unit of S-XIX, which was shown to be O-2-acetamido-2-deoxy-β-d-mannopyranosyl-(1→4)-O-α-d-glucopyranosyl-(1→2)-l-rhamnose 4′′-phosphate. Phosphoric acid forms a diester linkage in the S-XIX molecule, which explains the instability of S-XIX towards acid or alkali. The phosphodiester linkages in S-XIX join HO-1 of α-l-rhamnose and HO-4 of the 2-acetamido-2-deoxy-d-mannopyranosyl residue in the next repeating-unit. Treatment of S-XIX with alkali or alkaline-NaBH₄ produced the repeating units in a lower yield. The proposed structure of S-XIX is

     

Overestimates of the size of poly(A) segments.

Poly(A) molecules containing on average 25, 45 and 90 nucleotide residues are all eluted from DEAE-Sephadex in the presence of 7 M urea by approximately the same NaCl concentration which is higher than that required to elute 4 S and 5 S RNA. The same poly(A) molecules have electrophoretic mobilities on 12% polyacrylamide gels which are proportional to the logarithm of the number of nucleotide residues they contain but not to the number found in 4 S and 5 S RNA, even after denaturation of the RNA and performing electrophoresis in the presence of 2.2 M formaldehyde. As a result, many reported estimates of poly(A) size derived from such techniques are probably too large and need re-evaluation. Corrections are suggested for the use of 4 S and 5 S RNA as molecular weight markers for electrophoresis on 12% polyacrylamide gels.     

Decomposition reactions of amino sugars: the dehydration of 2-amino-2-deoxy-D-glucose

The stability of the title compound (1) was investigated at 100° in acidified aqueous solutions containing, in some instances, glycine or pyridine. In strong acid (3M hydrochloric acid), the sugar was relatively stable, and no identifiable decomposition-products were observed. In less-acidic solutions (≤0.5M hydrochloric acid) in the presence of glycine, substantial decomposition occurred with the production of 5-(hydroxymethyl)-2-furaldehyde (2) in 0.5-5.2% yield. The major dehydration products, however (up to 18% of the starting sugar), were pyrazine derivatives bearing dissimilar, four-carbon, acyclic-sugar side-chains attached to C-2 and C-5 of the ring, respectively, arising, most probably, from C-3-C-6 of the original sugar molecules. When the conversions were performed in deuterium oxide solution, carbon-bound isotope was observed in 2 (at the aldehyde carbon and at C-3) and, in the pyrazine derivatives, on the ring (positions 3 and 6), and on the sugar-derived, side-chains.     

Synthesis of N-(1-deoxyhexitol-1-yl)amino acids, reference compounds for the nonenzymic glycosylation of proteins

The N-(1-deoxy-D-mannitol-1-yl) and N-(1-deoxy-D-glucitol-1-yl) derivatives of L-valine, L-alanine, L-threonine, and L-leucine were prepared by reductive amination of D-mannose and D-glucose with the appropriate amino acids, in the presence of sodium cyanoborohydride. N epsilon-(1-Deoxy-D-mannitol-1-yl)- and N epsilon-(1-deoxy-D-glucitol-1-yl)-L-lysine were prepared by similar reactions of hexoses with N alpha-tert-butoxycarbonyl and N alpha-benzyloxycarbonyl-L-lysine, followed by removal of the protecting groups. The structures were confirmed by 1H-n.m.r. spectroscopy, which showed that each compound was completely free of its C-2 epimer. The synthetic compounds may be used as reference compounds for the identification of N-(1-deoxyhexitol-1-yl)amino acids formed when N-(1-deoxy-D-fructose-1-yl) groups of nonenzymically glycosylated proteins, of the hemoglobin A1c type, are reduced with sodium borohydride, and the protein is subjected to acid-catalyzed hydrolysis.     

Structural studies on a polysaccharide obtained from the cambium layer of a bael (Aegle marmelos) tree

The purified polysaccharide isolated from the cambium layer of a young bael (Aegle marmelos) tree contains galactose, arabinose, rhamnose, xylose, and glucose in the molar ratios of 10.0:9.8:1.4:1.9:1. Methylation analysis and Smith degradation studies established the linkages of the different monosaccharide residues. The anomeric configurations of the various sugar units were determined by oxidation of the acetylated polysaccharide with chromium(VI) trioxide. The oligosaccharides isolated from the polysaccharide by graded hydrolysis were characterized. The structural significance of these results is discussed.     

The dehalogenation of halouracils by hydroxylamine buffers.

At room temperature, hydroxylamine dehalogenates 5-Br-and 5-I-uracil. 5-Cl-uracil reacts to a much less extent. Reaction with 5-F-uracil yields the 6-hydroxyamino-adduct as a product. Kinetics monitored spectrally indicate that dehalogenation involves the formation of a 5-halo-6-hydroxyamino-5, 6-dihydrouracil intermediate which then slowly dehalogenates. 5-Bromo-6-methoxy-5,6-dihydrothymine, a model for the above intermediate, also dehalogenates yielding thymine as a product.Hydroxylamine (NH₂OH), a mutagenic agent (1,2) reacts with pyrimidine rings promoting such reactions as the formation of 5,6-dihydro-N⁴-hydroxy-6-hydroxyaminocytosine from cytosine (3,4) and both urea and isoxazoles from uracil derivatives (2,5,6). It is believed to be unreactive toward 5-substituted uracil derivatives (2,5,6) but has been reported to cause the dehalogenation of 5-bromouracil derivatives yielding Br^? and uracil as products (2,7,8). The object of this report is to demonstrate the generality of NH₂OH addition to the 5-halouracils with the subsequent dehalogenation of both 5-Br-and 5-I-uracil; reactions which appear to proceed via pathways similar to bisulfite buffer mediated halouracil dehalogenation (9–13). A preliminary report of this work has appeared (14).     

An improved preparation of 3-deoxy-d-erythro-hexos-2-ulose via the bis(benzoylhydrazone) and some related constitutional studies

Sugar osazones and glycosuloses rapidly and quantitatively react with hydroxylamine to produce oximes that give trimethylsilyl derivatives suitable for g.l.c. and mass spectral analysis. The reaction of d-glucose with benzoylhydrazine to give the bishydrazone of 3-deoxy-d-erythro-hexos-2-ulose (1) [H. El Khadem et al., Carbohydr. Res., 22 (1973) 381-89] was re-investigated, together with the conversion of this compound to the hexosulose. Although by-products are produced in the reaction, including the bis(benzoylhydrazone) (osazone) of d-glucose, the major product is the monohydrate of the bis(benzoylhydrazone) of 1 (colorless). The anhydrous (yellow) form can be prepared from the monohydrate by crystallization from absolute ethanol and has quite different physical properties. Improvements of the original preparation are described that allow the preparation of the bishydrazone and its subsequent conversion to 1via transhydrazonation in 44% overall yield, and with no detectable contamination by d-glucose or d-glucosone. Evidence is presented that the previously reported cyclic form of the bis(benzoylhydrazone) of d-glucose is the bis(benzoylhydrazone) (monohydrate) of 1.     

Circular dichroism and the conformational properties of soybean beta-amylase

The conformational properties of soybean β-amylase were investigated by the circular dichroism probe and measurement of enzyme activity. The enzyme exhibited a positive circular dichroism band at 192 nm, a negative band at 222 nm, and a shoulder near 210 nm. Analysis of the spectrum in the far ultraviolet zone indicated the presence of approximately 30% of α helix and 5–10% of β-pleated sheet, the rest of the polypeptide main chain possessing aperiodic structure. In the near ultraviolet reagion, the enzyme protein showed at least six positive peaks at 259, 265, 273, 281, 292, and 297 nm. The positive bands at 292 and 297 nm remained unaltered on acetylation of the enzyme by N-acetylimidazole and were assigned to tryptophanyl chromophores. These bands were affected in intensity in the presence of maltose or cycloheptaamylose, which indicates that some tryptophan residues are situated at the binding sites. The native conformation of soybean β-amylase was found to be sensitive to pH variation (below pH 5 and above pH 10), sodium dodecyl sulfate, guanidine hydrochloride, and heating to 50–55 °C. Complete disorganization of the secondary structure was attained by 6 m guanidine hydrochloride. Sodium dodecyl sulfate was effective in disturbing the tertiary structure of the enzyme but did not affect significantly the secondary structure. Enzymatic inactivation was paralleled by the decrease of circular dichroism bands in the near ultraviolet region as produced by the denaturants. It is concluded that the uniquely folded structure of the enzyme contains some less rigid domains and a rigid core stabilized by hydrophobic interactions, electrostatic interactions, and hydrogen bonds.     

Studies on 3-epimeric l-2-hexulose phenylosazones. Structure and anomeric configuration of the 3,6-anhydro-osazone derivatives obtained from l-xylo- and l-lyxo-2-hexulose phenylosazone

Dehydration of the 3-epimeric 2-hexulose phenylosazones l-xylo-hexulose phenylosazone and l-lyxo-hexulose phenylosazone afforded 3,6-anhydro-l-lyxo-2-hexulose phenylosazone (2) as the preponderant isomer from both. The identity of 2 was obtained by t.l.c., and by acetylation followed by comparison of the products. Acetylation of 2 with acetic anhydride-pyridine afforded the di-O-acetyl derivative 4, and further acetylation gave the N-acetyldi-O-acetyl derivative 5. Refluxing of 2 with copper sulfate afforded a C-nucleoside analog, namely, 2-phenyl-4-α-l-threofuranosyl-1,2,3-osotriazole (6). The anomeric configuration was determined by n.m.r. spectroscopy. The stereochemical course of the dehydration process and the mass spectra of compounds 2, 4, 5, and 6 are discussed.     

The structure of unit B-type glycopeptides from porcine thyroglobulin

The structure of Unit B-type glycopeptides (monosialo-type and disialo-type) was investigated by Smith degradation, methyllation, and mass spectral analysis. These glycopeptides contain three peripheral sugar chains. Two are composed of D-galactose residues linked at C-6 and 2-acetamido-2-deoxy-D-glucose residues linked at C-4, and the other is composed of a D-galactose residues linked at C-6, a 2-acetamido-2-deoxy-D-glucose residues linked at C-4, and a D-mannose residue linked at C-2. Most of these peripheral sugar chains are linked to two inner D-mannose residues which are substituted at C-3 and C-6, and constitute branching points. L-Fucose and N-acetyl-neuraminic acid residues are nonreducing terminal groups, and a di-N-acetylchitobiose moiety is linked to an asparagine residue in the peptide moiety. By methylation analysis of the oligosaccharide obtained by hydrazinolysis of the disialoglycopeptide, the L-fucose residues was found to be linked to C-6 of the 2-acetamido-2-deoxy-D-glucose residue linked to the asparagine residue. From these results, and from the previously reported data on the sugar sequence and the anomeric configurations of the linkages between sugar residues, structures for these glycopeptides are proposed.     

A high-resolution c.p.-m.a.s. 13C-n.m.r. study of solid-state cyclomaltohexaose inclusion-complexes: Chemical shifts and structure of the host cyclomaltohexaose

High-resolution, solid-state ¹³C-n.m.r. spectra were obtained for several crystalline cyclomaltohexaose inclusion-complexes. The resonances of C-1, C-4, and C-6 of the host were dispersed. The averaged ¹³C shifts of these resonances were in good agreement with the ¹³C shifts observed in solution, where the dispersion due to conformational diversity is expected to be averaged by rapid interconversion of the conformers. This result indicates that the most plausible source of the solid-state ¹³C-shift dispersions of the resonances of C-1 and C-4 is the diversity of conformations about the glycosidic linkage. The molecular origins of conformation-dependent ¹³C shifts are discussed.     

The interaction of Ca2+ with human factor IX

The binding isotherms of Ca²⁺ and Sr²⁺ to human blood coagulation Factor IX have been obtained at 25 °C and pH 7.4. In the case of both cations, a Scatchard plot of the data reveals that a single class of binding sites exist. For Ca²⁺, a total of 16.0 ± 1.0 sites, of K_D 7.3 ± 0.2 × 10^?4m, are present on human Factor IX. Similar analysis of the Sr²⁺ data indicates that Factor IX contains 11.0 ± 1.0 binding sites, with a K_D of 1.9 ± 0.1 × 10^?3m. Both Sr²⁺ and Mn²⁺ effectively displace Ca²⁺ from human Factor IX; whereas Mg²⁺ is considerably less potent in this regard. Conversely, Ca²⁺ is capable of nearly complete displacement of Sr²⁺ from its binding sites on human Factor IX. The activation of human Factor IX, by human Factor XIa, shows a complex dependence on the Ca²⁺ concentration. Sr²⁺ can substitute for Ca²⁺ in this activation process. Mn²⁺ cannot, in itself, substitute for Ca²⁺ in activation of Factor IX, but does significantly enhance the activation of Factor IX by Factor XIa at suboptimal levels of Ca²⁺. The rate of activation of human Factor IX by the coagulant protein of Russell's viper venom also shows a dependence on the presence of divalent cations. Here, however, a rigid specificity is not noted, since Ca²⁺, Sr²⁺, and Mn²⁺ all allow activation to proceed equally well.