Martin F. Chaplin

A rapid and sensitive method for the analysis of carbohydrate components in glycoproteins using gas-liquid chromatography☆

Abstract A rapid and simple gas-liquid chromatographic method for the determination of subnanomolar amounts of carbohydrates derived from glycoproteins is described. The procedure involves methanolysis in the presence of methyl acetate followed by removal of hydrogen chloride by coevaporation with t-butyl alcohol and trimethylsilylation. The method is also applicable to samples containing uronic acids and lipids.

Dietary fibre, physicochemical properties and their relationship to health.

Dietary carbohydrates that escape digestion and absorption in the small intestine include non- digestible oligosaccharides (carbohydrates with a degree of polymerisation between three and ten), resistant starch and non-starch polysaccharides. The physiological effects of this heterogeneous mixture of substrates are partly predictable on the basis of their physicochemical properties. Mono saccharide composition and chain conformation influence the rate and extent of fermentation. Water-holding capacity affects stool weight and intestinal transit time. Viscous polysaccharides can cause delayed gastric emptying and slower transit through the small bowel, resulting in the reduced rate of nutrient absorption. Polysaccharides with large hydrophobic surface areas have potentially important roles in the binding of bile acids, car cinogens and mutagens. Ispaghula is capable of binding bile acids through a large number of weak binding sites on the polysaccharide structure, and having greatest effect on the potentially more harmful secondary bile acids deoxycholic acid and lithocholic acid.

Fibre and water binding

The range of interactions between fibre and water and the consequential properties of the bound water are modelled and examined. Dietary fibre may interact with water by means of polar and hydrophobic interactions, hydrogen bonding and enclosure. The results of these interactions vary with the flexibility of the fibre surface. When the fibre is insoluble or junction zones are formed,this may result in profound changes in the surrounding water. Such interactions are capable of affecting the structuring and solvation properties of water well away from the immediate surfaces involved. In particular, the specific properties of water enclosed by dietary fibre are examined, an area of investigation previously receiving scant attention. The way this enclosure may affect the properties of water is exemplified by modelling the colon to show how fibre may exert a beneficial action by the preferential partitioning of hydrophobic carcinogens. Unfermented dietary fibre has a tendency to form low-density expanded water that acts as a preferential solvent for hydrophobic molecules when compared with the less-structured denser water within the much more hydrophilic mucus layer.

Purification and Partial Characterization of Tomato Extensin Peroxidase

Early plant defense response is characterized by elevation of activity of peroxidases and enhanced insolubilization of hydroxyproline-rich glycoproteins, such as extensin, in the cell wall. The insolubilization process (cross-linking between soluble extensin precursor molecules) is catalyzed by extensin peroxidases. We have ionically eluted extensin peroxidases from intact water-washed suspension-cultured tomato (hybrid of Lycopersicon esculentum Mill. and Lycopersicon peruvianum L. [Mill.]) cells and purified them to homogeneity by molecular sieve and cation-exchange chromatography. Four ionic forms of peroxidase (PI,PII,EPIII, and EPIV) were resolved; only the latter two cross-linked tomato soluble extensin. The molecular weight (34,000–37,000), amino acid composition, and isoelectric point (9.0) of the extensin peroxidases were determined. Substrate specificities of the enzymes were investigated: soluble extensin and potato lectin (a hydroxyproline-rich glycoprotein with a domain that strongly resembles extensin) were cross-linked by only two forms of the enzyme, whereas bovine serum albumin, aldolase, insulin, a number of other marker proteins, and proteins eluted from tomato cells (except extensin) could not be cross-linked. We have also isolated a yeast elicitor that enhances total peroxidase activity and extensin insolubilization within 1 h of challenge in cultured cells of tomato. A highly sensitive enzyme-linked immunosorbent assay technique using polyclonal antiserum raised against soluble tomato extensin was used to demonstrate extensin insolubilization in vivo. A tomato cell-wall peroxidase that cross-links extensin has been purified and may have a role in plant defense.

Primary structure of arabinoxylans of ispaghula husk and wheat bran.

The primary structures of ispaghula husk and wheat bran were investigated in order to determine how and why these fibres are among the most beneficial dietary fibres. To this end, the polysaccharide preparations have been subjected to enzymic hydrolysis and methylation analysis.The results have shown ispaghula husk and wheat bran to be very-highly-branched arabinoxylans consisting of linear f-D-(1-4)-linked xylopyranose (Xylp) backbones to which a-L-arabinofuranose (AraJ3 units are attached as side residues via a-(l13) and a-(1-02) linkages.Other substituents identified as present in wheat bran include P-D-glucuronic acid attached via the C(O)-2 position, and arabinose oligomers, consisting of two or more arabinofuranosyl residues linked via 1-2, 1-3, and 1-4 linkages. Ispaghula-husk arabinoxylan is more complex having additional side residues which include a-D-glucuronopyranose (GalAp)-(1-42)-linked-a-L-rhamnopyranose-(1-04)-0-D-Xylp, a-D-GalAp-(l-o3)-linked-a-L-Araf-(l-4)-[3-D-Xylp, and a-L-Araf-(l-43)-linked-P-D-Xylp-(1l -4)--D-Xylp. The beneficial effects of increased faecal bulk and water-holding capacity are undoubtedly related to the structures of the arabinoxylans, with differences in their efficacy to treat various functional bowel disorders due to their specific structural features.

Water: its importance to life

Textbooks increasingly include material concerning the importance of water but this topic is often treated over‐simplistically with insufficient attention being given to the central position of water in life processes. In this article, modern views of the fundamental role that water plays in biochemical function and process are summarized. The importance of water in the structures of nucleic acids and proteins is explained.

The putative common mechanism for inactivation of alkaline phosphatase isoenzymes

Abstract Alkaline phosphatase (E.C. 3.1.3.1) is a family of dimeric metalloenzymes with a complex inactivation mechanism that still remains to be elucidated. We have put forward a novel mechanism of Escherichia coli alkaline phosphatase inactivation, based on experimental as well as structural data for this isoenzyme. It suggests several stages of disruption of the intersubunit contact before the loss of enzyme activity. Here we present initial evidence that the mechanism could also be valid for mammalian isoenzymes. The evidence includes thermal inactivation kinetics and the structural similarity of different alkaline phosphatases inferred from the alignment of their amino acid sequences. The suggested inactivation mechanism of alkaline phosphatases is supported by recent experimental data showing an important role of three intersubunit contact areas in determining the stability of alkaline phosphatase isoenzymes.

Polypyrrole-Prussian blue films with controlled level of doping : codeposition of polypyrrole and Prussian blue

The search for a relatively inexpensive catalyst-electrode combination for electrochemical reactions arises from the importance of fuel cells and sensors. The discovery of polymer semiconductor films, such as polypyrrole [l], opened a new area in the field of the modification of electrode surfaces. Although polypyrrole itself is not a good electrocatalyst, its structure can be modified by a great number of compounds [2]. The redox activity of such species remains in polypyrrole, because of its semiconductor properties. Polypyrrole also serves as a suitable matrix for the immobilization of biological catalysts: enzymes [3,4] and whole cells [5].

Relative molecular mass of the polypeptide chain of βc-haemocyanin of Helix pomatia and carbohydrate composition of the functional units

1. 1. Sedimentation equilibrium at pH 9.25 of the subunit (twentieth molecules) of βc-haemocyanin of Helix pomatia at protein concentrations below 0.6 mg/ml yielded a Mw value of ⋍4.5 × 105, in good agreement with the Mr value (9 × 106) of the whole molecule. 2. 2. The sum of the Mr values of the tryptic fragments from sedimentation equilibrium and of the eight functional units from sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), amounted to 4.6 × 105. 3. 3. Carbohydrate is unevenly distributed over the functional units: g >b >e >a >h >d >f >c. On subtracting its weight contribution (8.25%) a Mr of ⋍4.2 × 105 was obtained for the polypeptide part of the subunit. 4. 4. The βc-haemocyanin subunit and some of its proteolytic fragments are proposed as markers for SDS-PAGE in the high-Mr range.

Water’s Hydrogen Bond Strength

Water is necessary both for the evolution of life and its continuance. It possesses particular properties that cannot be found in other materials and that are required for life-giving processes. These properties are brought about by the hydrogen bonded environment particularly evident in liquid water. Each liquid water molecule is involved in about four hydrogen bonds with strengths considerably less than covalent bonds but considerably greater than the natural thermal energy. These hydrogen bonds are roughly tetrahedrally arranged such that when strongly formed the local clustering expands, decreasing the density. Such low density structuring naturally occurs at low and supercooled temperatures and gives rise to many physical and chemical properties that evidence the particular uniqueness of liquid water. If aqueous hydrogen bonds were actually somewhat stronger then water would behave similar to a glass, whereas if they were weaker then water would be a gas and only exist as a liquid at sub-zero temperatures. The overall conclusion of this investigation is that waters hydrogen bond strength is poised centrally within a narrow window of its suitability for life.