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Glyoxylic acid, added to aqueous suspensions of chitosan, causes immediate dissolution of chitosan and gel formation within 3–4 h if the pH is 4.5–5.5. Solutions at lower pH values gel after 2 min of warming at 60–80°. Chitosan glyoxylate solutions brought to alkaline pH with sodium hydroxide do not precipitate chitosan. Evidence is given that a Schiff base, namely N -(carboxymethylidene)chitosan, is formed. N -(Carboxymethylidene)chitosans are reduced by sodium cyanoborohydride at room temperature to give N -(carboxymethyl)chitosans, obtained as white, free-flowing powders, soluble in water at all pH values. A series of N -(carboxymethyl)chitosans having various degrees of acetylation and N -carboxymethylation was obtained, and characterized by viscometry, elemental analysis, and i.r. spectrometry. For the fully substituted N -(carboxymethyl)chitosans, the pK′ is 2.3, the pK″ is 6.6, and the isoelectric point is 4.1. The addition of N -(carboxymethyl)chitosan to solutions (0.2–0.5m m ) of transition-metal ions produces immediate insolubilization of N -(carboxymethyl)chitosan-metal ion chelates.

Ascorbic acid immediately dissolves Euphausia superba chitosan upon mixing and forms chitosan ascorbate; during the 6-h period after dissolution in water at pH 5–7, ascorbate is oxidized to dehydroascorbate which undergoes Schiff reaction with the amino groups of chitosan, thus yielding a viscous solution of a polymeric ketimine. The latter is characterized by infrared spectrometry, circular dichroism spectropolarimetry, viscometry and alkalimetry. When brought into contact with transition metal ions, the chitosan ascorbate ketimine yields insoluble metal chelates. Upon reduction with sodium cyanoborohydride, the water-insoluble N-[2-(1,2-dihydroxyethyl)tetrahydrofuryl] chitosan (NDTC) is obtained, which shows enhanced capacity for uranium, up to 800 mg U/g from solutions at pH 4·5.

Chitins and chitosans from crabs and shrimps as well as the chitosan-glucan complex from Aspergillus niger show an ESR singlet at 3387–3391 G and g values 2.00117-200354; this signal is altered by the action of oxygen from the atmosphere and from hydrogen peroxide, and by hot water.

The imine formed by chitosan with phthalaldehydic acid was reduced with sodium cyanoborohydride and the resulting N -( o -carboxybenzyl) chitosan (NCBC) was insolubilised with ethanol and acetone and obtained as a white, free-flowing powder, soluble in both acidic and alkaline solutions. A sample of NCBC with the following degrees of substitution: acetamido 42%±4%, N -( o -carboxybenzyl) amine 43%±3%, free amine 15%±1% and containing 16%±1% moisture, was characterised by IR and UV-Vis. spectrometry, titration and viscometry. The isoelectric point was 6·8; the pK a values were 5·7 and 8·0. NCBC could be determined by UV-Vis. spectrophotometry in aqueous solutions at 274 nm; maximum viscosity of the solutions was observed at pH 4. Upon addition of NCBC to transition metal ion solutions (0·1–0·5 m m ) chelation and insolubilisation took place immediately. The dependence of the collection percentage on pH, NCBC and metal ion concentrations was studied for nine metal ions.

Chitosan was N-permethylated by reaction with formaldehyde and sodium borohydride under controlled conditions (pH 4·0, 15°C, reaction times 12 and 8 h, respectively). The N-permethylated chitosan was reacted with methyl iodide at 35°C and N-trimethyl chitosan iodide with a quaternary nitrogen degree of 60% was obtained. This material may have uses as an antibiotic and an ion exchange material.

FT-IR analysis shows that treatment of F1ATPase with the inhibitors DCCD and Nbf-Cl, in the presence of saturating concentrations of ADP and AMP-PNP and in the absence of Mg2+, does not modify the secondary structure of the enzyme, but significantly modifies its compactness and thermal stability, although to different extents. Nbf-Cl causes a significant increase in stabilisation, in addition to that induced by nucleotides, while DCCD is less effective in this regard. Determination by HPLC of the exchange rate, in the absence of Mg2+, of tightly bound nucleotides of F1ATPase treated with the two inhibitors shows that DCCD does not significantly affect the exchange rate of ADP with AMP-PNP and vice versa in catalytic and non-catalytic tight sites, while Nbf-Cl selectively reduces the enzyme's capacity to exchange ADP bound in the tight catalytic site. It is suggested that the effects of DCCD, unlike those of Nbf-Cl, are closely related to the presence or absence of Mg2+.

The secondary structure of delipidated and egg phosphatidylcholine or asolectin reconstituted mitochondrial ATP synthase complex from beef heart was investigated by Fourier transform infrared spectroscopy. Upon reconstitution, the infrared spectra of ATP synthase revealed an increase in turns and a concomitant decrease in β-sheet content which occurred to a larger extent in the presence of asolectin rather than in the presence of egg phosphatidylcholine. These data correlate with kinetic data showing a higher ATPase activity of the asolectin reconstituted enzyme protein than the egg phosphatidylcholine reconstituted or delipidated enzyme complexes.

Gas chromatography of a number of amines, alcohols and sulfur derivatives was carried out on chitin and partially deacetylated chitins as well as on chitosan. The retention time of methanol is proportional to the degree of acetylation, and therefore a method is proposed for the gas-chromatographic determination of the degree of acetylation of chitin/chitosan. The analysis of the infrared spectra of chitin/chitosan also permits one to determine the degree of acetylation by using the ratio of the bands 1550 and 2878 cm−1.

Permeability of liposomes made from mixtures of unoxidized and singlet oxygen oxidized phosphatidylcholine has been related to the degree of lipid oxidation expressed as hydroperoxide moiety content in the lipids. The effect of oxidation on the liposomes permeability has been studied by fluorometry using calcein as a fluorescent probe that undergoes self quenching when highly concentrated inside liposomes. The liposomes containing 73 % and 5% of hydroperoxides retain respectively 64.5 and 96.3 % of calcein with respect to that retained by the liposomes made from unoxidized phosphatidylcholine. The fluorescence data show a linear relationship between the liposome permeability and the oxidation degree of lipids.

Multilamellar liposomes, from mixtures of unoxidized (control) and singlet oxygen oxidized phosphatidylcholine, were studied by steady-state fluorescence anisotropy and multifrequency phase fluorometry using 1,6-diphenyl-1,3,5-hexatriene (DPH) as fluorescent probe. Lifetime fluorescence decay of the DPH-labeled liposomes was analyzed either by a model of discrete exponential components and a model that assumes a continuous distribution of lifetime values. Increasing the oxidized phosphatidylcholine content in the liposomes, an increase of the membrane interior polarity and a decrease of membrane fluidity occurs which can be related to the hydroperoxide-lipids and double bonds conjugation, respectively.

Hsp70 (DnaK) is a highly conserved molecular chaperone present in bacteria, eukaryotes, and some archaea. In a previous work we demonstrated that DnaK from the archaeon Methanosarcina mazei (DnaK(Mm)) and the DnaK from the bacterium Escherichia coli (DnaK(Ec)) were functionally similar when assayed in vitro but DnaK(Mm) failed to substitute for DnaK(Ec) in vivo. Searching for the molecular basis of the observed DnaK species specificity we compared substrate binding by DnaK(Mm) and DnaK(Ec). DnaK(Mm) showed a lower affinity for the model peptide (a-CALLQSRLLS) compared to DnaK(Ec). Furthermore, it was unable to negatively regulate the E. coli sigma32 transcription factor level under heat shock conditions and poorly bound purified sigma32, which is a native substrate of DnaK(Ec). These observations taken together indicate differences in substrate specificity of archaeal and bacterial DnaKs. Structural modeling of DnaK(Mm) showed some structural differences in the substrate-binding domains of DnaK(Mm) and DnaK(Ec), which may be responsible, at least partially, for the differences in peptide binding. Size-exclusion chromatography and native gel electrophoresis revealed that DnaK(Mm) was found preferably in high molecular mass oligomeric forms, contrary to DnaK(Ec). Oligomers of DnaK(Mm) could be dissociated in the presence of ATP and a substrate (peptide) but not ADP, which may suggest that monomer is the active form of DnaK(Mm).

Hsp70s are chaperone proteins that are conserved in evolution and present in all prokaryotic and eukaryotic organisms. In the archaea, which form a distinct kingdom, the Hsp70 chaperones have been found in some species only, including Methanosarcina mazei. Both the bacterial and archaeal Hsp70(DnaK) chaperones cooperate with a GrpE co-chaperone which stimulates the ATPase activity of the DnaK protein. It is currently believed that the archaeal Hsp70 system was obtained by the lateral transfer of chaperone genes from bacteria. Our previous finding that the DnaK and GrpE proteins of M. mazei can functionally cooperate with the Escherichia coli GrpE and DnaK supported this hypothesis. However, the cooperation was surprising, considering the very low identity of the GrpE proteins (26%) and the relatively low identity of the DnaK proteins (56%). The aim of this work was to investigate the molecular basis of the observed interspecies chaperone interaction. Infrared resolution-enhanced spectra of the M. mazei and E. coli DnaK proteins were almost identical, indicating high similarity of their secondary structures, however, some small differences in band position and in the intensity of amide I' band components were observed and discussed. Profiles of thermal denaturation of both proteins were similar, although they indicated a higher thermostability of the M. mazei DnaK compared to the E. coli DnaK. Electrophoresis under non-denaturing conditions demonstrated that purified DnaK and GrpE of E. coli and M. mazei formed mixed complexes. Protein modeling revealed high similarity of the 3-dimensional structures of the archaeal and bacterial DnaK and GrpE proteins.

Waste mycelia of Aspergillus niger from a citric acid production plant are simply treated with boiling 30-40% NaOH aqueous solutions for 4-6 hr to obtain the insoluble chitosan-glucan complex whose infrared, ESR, and x-ray diffraction spectra are reported. A number of transition- and post-transition-metal ions are chelated and collected by chitosan-glucan with higher yields than by animal chitosan. Immediate flocculation occurs upon mixing chitosan-glucan dispersions with alginate and polymolybdate solutions. Membranes are also obtained from chitosan-glucan dispersions in acetic acid or in chloral and dimethyl formamide mixtures.

Aspartate glucan, glycine glucan, and serine glucan obtained by reductive amination of oxalacetic acid, glyoxylic acid, and ..beta..-hydroxypyruvic acid, respectively, with polyglucosamine were tested as chromatographic chelating media. Crosslinked glycine glucan exhibited high capacities for cobalt and copper, even in acidic solutions (pH 2.9). Breakthrough points for 10 mg/L solutions through 6.0 x 0.6 cm columns containing 200 mg of polymer were at 1.8 L for both ions; for 1 mg/L solutions, they were at 4.0 and 12.0 L. for cobalt and copper, respectively. Crosslinked glycine glucan could remove microgram amounts of cobalt and copper from fluoride and chloride brines. Cobalt and copper could be separated by elution with 0.25M sulfuric acid.