Immunological relevance of malonic dialdehyde I. Preparation of Schiff''s bases from lysozyme or polylysine reacted with malonic dialdehyde

Malonic dialdehyde (MDA) is produced in all mammalian tissues either as an end product of lipid peroxidation or as a by-product of arachidonic acid metabolism. It may either be quickly oxidized to carbon dioxide or combine covalently with primary amino groups of proteins, phospholipids or nucleic acids. In the latter case, fluorescent Schiff's bases with 1-amino-3-iminopropene (AIP) bridges are produced. MDA metabolism is now fairly well elucidated, while that of MDA-cross-linked biological molecules remains unknown. Aiming at investigating the fate of such cross-linked molecules in mammalian organisms, and their biological relevance, we tried in the present study to prepare reproducibly Schiff's bases from chicken egg white lysozyme reacted with MDA. The resulting mixture of different Schiff's bases (ML) was fractionated into single oligomeric fractions by gel-filtration chromatography. ML and the single oligomeric fractions obtained from this mixture were controlled by fluorescence measurements for their content of AIP bridges, and by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) for their content of different oligomers. ML contained monomers, dimers, trimers and other oligomers, as shown by SDS-PAGE. The corresponding single oligomeric fractions were satisfactorily separated by gel-filtration chromatography (purity better than 94%, as determined by SDS-PAGE). Schiff's bases from poly-L-lysine reacted with MDA (MP) were also prepared. Their fluorescence emission spectrum was similar to that of ML and to that of the single oligomeric fractions obtained from ML.     

双醛淀粉的制备与应用

介绍了双醛淀粉优良的物化、生化性能,及其在近二十年来被广泛应用的状况,如应用于蛋白质化学、酶的固定化、药物化学等领域.提出以拟均相体系制备双醛淀粉的方法,反应条件温和,可提高氧化速度,可改善产品性能。     

Stabilization of type I collagen using dialdehyde cellulose

Type I collagen from rat tail tendon (RTT) fibres was crosslinked with dialdehyde cellulose to bring about stabilization of the matrix. Dialdehyde cellulose (DAC) was prepared by periodate oxidation of hydrolyzed cellulose. Autoclaving of DAC resulted in hydrolysis and lower molecular weight oligomeric species. The formation of the crosslinked network between DAC and the collagen fibres has brought about significant thermal and enzymatic stability to collagen. DAC crosslinked collagen fibres exhibited an increase in hydrothermal stability by 20 °C with autoclaved DAC at pH 8. The collagen matrix resulted in an increase in denaturation peak temperature (T_D) and an increase in phase change of activation energy (E_a) and enthalpy change (ΔH) for the shinking process indicating intermolecular crosslinking arising from covalent interactions. Thermal stability and crosslinking efficiency was found to increase with pH and concentration of DAC. DAC treated collagen exhibited 93% resistance to collagenolytic hydrolysis.     

Covalent immobilization of a glucoamylase to bagasse dialdehyde cellulose

Cellulose fibres from bagasse were oxidized by sodium periodate in sulphuric acid media at positions 2 and 3 of the anhydroglucose unit to produce dialdehyde cellulose. The aldehyde groups of the dialdehyde cellulose were able to react with amino groups of a glucoamylase to form covalent bonds and result in a dialdehyde cellulose immobilized enzyme. The optimum pH of this immobilized enzyme and free enzyme were in the range of 3.0–5.0 and 3.5–5.0, respectively. The optimum temperature for both the free and immobilized enzymes was 60–65 °C. The relative remaining activity of the immobilized enzyme was 36% and its stability was very good, since it could be reused for over 30 cycles. Its activity decreased from the first to the seventh reuse cycles, due to the slow detachment of non-covalently bound enzyme. However, activity tended to stabilize after the seventh cycle of reuse, indicating very stable covalent binding between the enzyme and dialdehyde cellulose.     

双醛淀粉柔性固定木瓜蛋白酶研究

提出“柔性固定化酶”的模型,即：用一亲水、柔性高分子链接枝于载体表面制得柔性固定化载体,再用其以共价键合的方式进行酶的柔性固定化。其特点是：柔性固定可改善因直接固定化及手臂固定化使酶失活的缺陷,并提高固定化酶的自由度;如选用粒径单分散微球可改善固定化反应及固定化酶催化反应的均一性。以双醛淀粉(DAS)为柔性链对羧基化聚苯乙烯载体进行柔性化修饰后,固定木瓜蛋白酶,其活力回收率可达50％．相当于用戊二醛进行手臂固定化的活力回收率的2倍。     

Formation of nanometal particles in the dialdehyde starch matrix

Environmentally friendly method of the preparation of dialdehyde starch (DAS) based composites containing nanosilver (DAS/Ag) and nanogold (DAS/Au) as reducing and protecting agents was developed. UV–vis spectroscopy, transmission electron microscopy (TEM) and X-ray diffraction (XRD) confirmed formation of about 10 nm ball shaped Ag and Au nanoparticles situated within the polysaccharide template. Thermal properties of the composites were characterized involving differential scanning calorimetry (DSC), whereas molecular weights of polysaccharide chains of the matrix were estimated with the size exclusion chromatography coupled with multiangle laser light scattering and refractometric detectors (HPSEC-MALLS-RI).     

Synthesis and properties of carrier-fixed enzymes. VII. Linking of glucoamylase to dialdehyde cellulose, carboxymethylcellulose hydrazide and carboxymethylcellulose azide]

Glucoamylase (alpha-1,4-glucan glucohydrolase, EC 3.2.1.3) has been covalently linked to dialdehyde cellulose resulting in an immobilized enzyme containing 0.98% protein and an activity of 4.5 mg of the native enzyme per g of matrix, i.e. 46% relative activity. The complex lost its activity in continuous and batch hydrolysis of starch at 55 degrees C down to a limit of 18% of its original value. In contrast, the activity of the complex did not change when working at a temperature of 25 degrees C. Glucoamylase-carboxymethylcellulose complexes synthesized via carboxymethylcellulose hydrazide and azide, in contrast to MAEDA und SUZUKI [1], showed only an activity of 1 mg of the native enzyme per g of matrix. We did not succeed in coupling periodate-oxidized glucoamylase to carboxymethylcellulose hydrazide because the enzyme used lost nearly all of its activity already during periodate oxidation.     

Effect of rheopolyglucin and dialdehyde dextran on thermostability of human haemoglobin molecules

Structural characteristics and thermostability of human haemoglobin molecules, modified by reopolyglukine and dialdehyddextrane (DAD) have been studied. Haemoglobin mainly preserves its oxyform when including reopolyglukine in mixture (the mol. ratio of protein to dextrane was 1:4), and disordering of polypeptide chains and increasing the volume of haemoproteids molecules are noticed. Assessment of the influence of mixture pH, exposure and temperature of incubation and the rate of dextrane oxidation on the intensity of the process of human haemoglobin conjugation with DAD have been chosen. Formation of the haemoglobin-DAD complex leads to shielding protein chromophoral groups by polysugar matrix and to transforming the part of haemoproteid molecules from lowspin form (HbO2) to highspin forms (Hb, MetHb). It has been detected that temperature of denaturational points for native protein and haemoglobin in the presence of reopoliglukine is 60 degrees C, but it is 80 degrees C for the haemoglobin-DAD conjugate. The latter is probably determined by increasing hydrophobic interactions inside the protein globule under the effect of DAD and by ability of the surface-bound carbohydrate components prevent association of haemoglobin molecules.     

Novel carotenoid oxidase involved in biosynthesis of 4,4'-diapolycopene dialdehyde

Biosynthesis of C(30) carotenoids is relatively restricted in nature but has been described in Staphylococcus and in methylotrophic bacteria. We report here identification of a novel gene (crtNb) involved in conversion of 4,4'-diapolycopene to 4,4'-diapolycopene aldehyde. An aldehyde dehydrogenase gene (ald) responsible for the subsequent oxidation of 4,4'-diapolycopene aldehyde to 4,4'-diapolycopene acid was also identified in Methylomonas. CrtNb has significant sequence homology with diapophytoene desaturases (CrtN). However, data from knockout of crtNb and expression of crtNb in Escherichia coli indicated that CrtNb is not a desaturase but rather a novel carotenoid oxidase catalyzing oxidation of the terminal methyl group(s) of 4,4'-diaponeurosporene and 4,4'-diapolycopene to the corresponding terminal aldehyde. It has moderate to low activity on neurosporene and lycopene and no activity on beta-carotene or zeta-carotene. Using a combination of C(30) carotenoid synthesis genes from Staphylococcus and Methylomonas, 4,4'-diapolycopene dialdehyde was produced in E. coli as the predominant carotenoid. This C30 dialdehyde is a dark-reddish purple pigment that may have potential uses in foods and cosmetics.     

Galactose dialdehyde as potential protein cross-linker: proof of principle

Combined enzymatic oxidation of D-galactose by D-galactose oxidase [EC 1.1.3.9] in water, amination with butylamine, and oxalic acid catalyzed Amadori rearrangement in methanol yielded 1,6-bis(butylamino)-1,6-dideoxy-erythro-hexo-2,5-diulose, demonstrating how in situ formed galacto-hexodialdose can be used to cross-link protein residues. The various species formed during this three-step conversion are present as bicyclic structures in solution as established by 13C labeling and in situ NMR spectroscopy of the reaction mixtures. Using protein (gelatin) instead of butylamine, distinct Amadori product formation was observed using 99% enriched D-(1-(13)C)- and D-(2-(13)C)-galactose.     

DNA duplexes with reactive dialdehyde groups as novel reagents for cross-linking to restriction- modification enzymes.

To create new, effective reagents for affinity modification of restriction-modification (R-M) enzymes, a regioselective method for reactive dialdehyde group incorporation into oligonucleotides, based on insertion of a 1-beta-D-galactopyranosylthymine residue, has been developed. We synthesized DNA duplex analogs of the substrates of the Eco RII and Mva I R-M enzymes that contained a galactose or periodate-oxidized galactose residue as single substituents either in the center of the Eco RII (Mva I) recognition site or in the flanking nucleotide sequence. The dependence of binding, cleavage and methylation of these substrate analogs on the modified sugar location in the duplex was determined. Cross-linking of the reagents to the enzymes under different conditions was examined. M. Eco RII covalent attachment to periodate-oxidized substrate analogs proceeded in a specific way and to a large extent depended on the location of the reactive dialdehyde group in the substrate. The yield of covalent attachment to a DNA duplex with a dialdehyde group in the flanking sequence with Eco RII or Mva I methylases was 9-20% and did not exceed 4% for R. Eco RII.     

Mechanism of formation of malonic dialdehyde during liposome interaction with cells

In the presence of intact Ehrlich ascite carcinoma cells and the supernatant obtained by preincubation and subsequent precipitation of cells, egg phosphatidylcholine is oxidized in liposomes to form malonic dialdehyde (MDA). Catalase and carbon dioxide markedly reduce, whereas sodium azide increases MDA accumulation during liposome incubation with the cells. EDTA, diethylthiocarbonate and alpha-tocopherol effectively inhibit, whereas ascorbate and cysteine strongly activate MDA synthesis in both cases. Superoxide dismutase has no appreciable effect on these processes. It is concluded that metal-containing catalysts and the H2O2 released by intact cells into the incubation medium induce lipid peroxidation in liposomes.     

S-Adenosyl-L-homocysteine dialdehyde: An affinity labeling reagent for histamine-N-methyltransferase

S-Adenosyl-L-homocysteine (SAH) was converted to 2′-O-[(R)-formyl(adenin-9-yl)methyl]-3′-S-homocysteinyl-3′-deoxy-(R)-glyceraldehyde (SAH dialdehyde) by periodic acid oxidation. SAH dialdehyde was then reduced with sodium borohydride to the corresponding diol, 2′,3′-acyclic SAH. SAH dialdehyde, but not 2′,3′-acyclic SAH, was found to inhibit histamine-N-methyltransferase (HMT). Neither analog showed significant inhibitory activity toward other methyltransferases. The inhibition of HMT by SAH dialdehyde was irreversible with the inactivation following first-order kinetics. A kinetic analysis suggests the formation of a dissociable enzyme-inhibitor complex prior to inactivation. The enzyme could be protected from inactivation by inclusion of S-adenosyl-L-methionine in the preincubation mixture.