Enzymatic generation of binary block-copolymeric structures: mathematical analysis based on triad frequencies evaluated by NMR.

A mathematical model is derived for describing a multiple-attack pathway for enzymatic generation of block structure in binary linear copolymers having initially a randomized sequential structure. The model is based on sequential information in terms of copolymer monads, diads, and triads estimated by nmr spectroscopy, and is applicable to enzymes attacking next to a reacted unit in the polymer chains. Then the block distribution of unreacted units remains constant and explicit relationships are provided. The probability of triad frequencies as a function of monads, i.e., progress curve of enzyme copolymer sequential structure, allows us to characterize the enzymatic mode of attack independently of enzyme kinetics. The produced fractions of heterogeneous triads centered by reacted units are shown to be affected, to a large extent, by the degree of multiple attack (d) entering into the formula as a variable parameter. The single-chain, d = infinity, and multiple-chain mechanisms, d = 1, representing the two extremes of the treated mechanism, are very clearly discriminated.

Quantitative studies of the non-productive binding of lysozyme to partially N-acetylated chitosans. Binding of large ligands to a one-dimensional binary lattice studied by a modified McGhee and von Hippel model.

This paper considers the non-productive (inhibitory) binding of chitosans to lysozyme from chicken egg white. Chitosans are linear, binary heteropolysaccharides consisting of 2-acetamido-2-deoxy-beta-D-glucose (GlcNAc; A-unit) and 2-amino-2-deoxy-beta-D-glucose (GlcN, D-unit). The active site cleft of lysozyme can bind six consecutive sugar residues in subsites named A-F, and specific binding of chitosan sequences to lysozyme occurs with A-units in subsite C. Chitosans with different fractions of A-units (FA) induced nearly identical changes in the 1H NMR spectrum of lysozyme upon binding, and the concentration of bound lysozyme could be determined. The data were analysed using a modified version of the McGhee and von Hippel model for binding of large ligands to one-dimensional homogeneous lattices. The average value of the dissociation constant for different sequences that may bind to lysozyme (K(ave)D) was estimated, as well as the number of chitosan units covered by lysozyme upon binding. K(ave)D decreased with increasing FA-values at pH 3 and 4.5, while the opposite was true at pH 5.5. Contributions from different hexamer sequences to K(ave)D of the chitosans were considered, and the data revealed interesting features with respect to binding of lysozyme to partially N-acetylated chitosans. The relevance of the present data with respect to understanding lysozyme degradation kinetics of chitosans is discussed.

The interactions between highly de-N-acetylated chitosans and lysozyme from chicken egg white studied by 1H-NMR spectroscopy.

We have investigated the binding interactions between highly de-N-acetylated chitosans and lysozyme from chicken egg white by one-dimensional and two-dimensional 1H-NMR spectroscopy. A fully de-N-acetylated chitosan (fraction of N-acetylated units, F < 0.001) induced no observable changes in the 1H chemical shifts of lysozyme. However, a chitosan with F(A) = 0.04, where the N-acetylated units are predominantly surrounded by de-N-acetylated units (a monoacetylated sequence), induced significant shifts of several lysozyme resonances, demonstrating a specific interaction between lysozyme and de-N-acetylated units in the chitosan. The interaction between the two positively charged molecules increased with increasing ionic strength, as expected. The dissociation constant (Kd) between lysozyme and the monoacetylated sequence was strongly dependent on pH* (pH measured in D2O), with Kd = 0.02+/-0.01 mM at pH* 6.0, Kd = 0.11+/-0.02 mM at pH* 4.5, and Kd approximately 2 mM at pH* 3, suggesting that electrostatic forces contribute to the observed binding. The complex was strikingly stable, with bound lifetimes in the range of 10-25 ms at pH* 4.5 and 328-300 K. Most lysozyme resonances that were affected by the binding were assigned, and we suggest that the monoacetylated chitosan sequence binds to the active site cleft of lysozyme with the N-acetylated unit in subsite C. Assuming this binding mode, we have discussed the contributions in energetic terms from individual subsites of lysozyme towards binding of N-acetylated and de-N-acetylated units.

A new NMR airlift bioreactor used in 31P-NMR studies of itaconic acid producing Aspergillus terreus.

An airlift bioreactor for in-vivo NMR studies of cells is described. The 10-mm diameter airlift reactor was constructed for studies of mycelial/pellet forming organisms, grown in suspension. With this device 161 MHz 31P-NMR spectra of living Aspergillus terreus cells, producing itaconic acid, have been obtained. Signals were observed for intra- and extracellular orthophosphate, glycerol-3-phosphorylethanolamine (GPE), glycerol-3-phosphorylcholine (GPC), sugar phosphates and polyphosphate. The spectra also showed broad overlapping resonances in the shift range of NAD(H) and NADP(H). Polyphosphate disappeared when the respiratory gas was exchanged for pure N2. The intracellular pH was estimated at 6.2. In spectra of cell extracts approx. 60 peaks were observed in the range of 20 to -22 ppm, and they confirmed the appearance of the metabolites observed in living cells.

Evidence for double helical kappa-carrageenan in aqueous LiI-solution and model for iodide binding.

NMR experiments with a 21412 Redfield observation pulse were carried out on a kappa-carrageenan aqueous solution containing 0.5M LiI. A detected hydrogen-bonded proton resonance at approximately 17.6 ppm, is in accordance with an ordered carrageenan structure in the solution similar to the double, parallel-stranded helix with interstrand hydrogen-bonds found in the condensed state by R.P. Millane, R. Chandrasekaran, S. Arnott, and I.C.M. Dea (Carbohydr. Res. 198, 1 1988). Starting with the published iota-carrageenan coordinates obtained from the Brookhaven Databank, we modelled this kappa-carrageenan structure by the molecular modelling software INSIGHTH-II. The kappa-carrageenan double-helix has a wide interior that can accommodate iodide. Conversely, the iota-carrageenan double-helix has a narrow interior not suited for a large ion like iodide. We propose that the detected selective iodide binding sites in the ordered kappa-carrageenan structure (H. Grasdalen, and O. Smidsrød, Macromolecules 14, 1842 1981) could very well be in the interior of the double-helix.

The use of neocarrabiose oligosaccharides with different length and sulphate substitution as model compounds for 1H-NMR spectroscopy.

The high-field 1H-NMR spectra of various carrageenan oligosaccharides at room temperature are given. The assignments were faciliated by the use of proton double-quantum coherence (DQCOSY) and 1H-13C chemical shift correlation 2D NMR spectroscopy, and by comparing high-field 1H-NMR spectra of various 4-sulphated oligosaccharides of the neocarrabiose type. The effects of anomeric configuration on the 1H resonances on the same or neighbouring units are discussed. The 13C-NMR shift data are given for the tetrasaccharide of kappa-carrageenan.

13C-n.m.r. studies of the acetylation sequences in partially N-deacetylated chitins (chitosans).

Chitosans obtained under homogeneous conditions of N-deacetylation, with degrees of N-deacetylation between 46% and 94%, were depolymerised and their 125-MHz 13C-n.m.r. spectra have been interpreted. The sequence of 2-acetamido-2-deoxy-beta-D-glucopyranose (GlcNAc) and 2-amino-2-deoxy-beta-D-glucopyranose (GlcN) residues influenced the chemical shifts, and the diad and triad frequencies have been calculated. Chitosans that were N-deacetylated under homogeneous and heterogeneous conditions gave values for the diad and triad frequencies that were consistent with a random arrangement of GlcN and GlcNAc residues.

Determination of the degree of N-acetylation and the distribution of N-acetyl groups in partially N-deacetylated chitins (chitosans) by high-field n.m.r. spectroscopy.

The composition and sequence of 2-acetamido-2-deoxy-beta-D-glucose (GlcNAc) and 2-amino-2-deoxy-beta-D-glucose (GlcN) residues in partially N-deacetylated chitosans, prepared under homogeneous and heterogeneous conditions, have been determined by 1H-n.m.r. spectroscopy. It was necessary to depolymerise the chitosan slightly by treatment with nitrous acid before spectroscopy. A sequence-dependent deshielding of H-1 of the GlcNAc residues made it possible to determine the proportions of the four possible diads. Chitosan prepared by N-deacetylation under homogeneous conditions gave values for the diad frequencies that were roughly consistent with a random distribution of the N-acetyl groups. Samples prepared under heterogeneous conditions have a frequency of the GlcNAc-GlcNAc diad slightly higher than for a random (Bernoullian) distribution. The chitosans, prepared under both homogeneous and heterogeneous conditions, with a degree of acetylation of 50% were soluble at neutral pH.

Monomer sequence and acetylation pattern in some bacterial alginates.

The sequential structures and acetylation patterns of alginates from several strains of Azotobacter vinelandii and Pseudomonas species, including P. aeruginosa, P. putida, P. fluorescens, and P. mendocina, have been studied by 1H-n.m.r. spectroscopy. O-Acetyl groups were exclusively associated with the D-mannuronic acid residues and the degree of acetylation varied in the range 4-57%, depending upon the proportion of this acid in the polymer. 1H-N.m.r. spectroscopy of a naturally occurring and an artificially acetylated D-mannuronan made it possible to determine the degrees of acetylation at O-2, O-3, and O-2,3. The most conspicuous difference between alginates from A. vinelandii and the four Pseudomonas species was the complete absence of consecutive L-guluronic acid residues in the latter.

31P-NMR studies of phosphate metabolites in intact red and white swimming muscles of cod (Gadus morhua L.).

31P-NMR was used to characterise intracellular phosphate pools and their post mortem changes at 7 degrees C in intact red and white cod muscles under anaerobic conditions. A total phosphate content of 55 and 60 mM was observed in red and white muscle, respectively. The concentration of P-creatine was 14 mM in the white and 9 mM in the red muscle, while that of inorganic phosphate, Pi (30 mM), ATP (9 mM), and sugar phosphate (5 mM) were similar in both muscles. During the first 90 min after death, the decrease in P-creatine showed a first order breakdown with a concomitant stoichiometric increase in Pi content, whereas the ATP and sugar phosphate remained the same. The intracellular pH decreased from 7.4 to 7.3 in this period. The steady-state rate constant of myosin ATPase was 0.0054 and 0.0022/min for red and white muscles, respectively. Individuals kept under diminished oxygen tension prior to being killed, showed a reduced P-creatine level in both muscles.

Trialkyl phosphorothioates and glutathione S-transferases.

Using a rat liver cytosol source of enzyme trialkyl phosphorothioates have been shown to be substrates of glutathione S-transferases. Using OSS-trimethyl phosphorodithioate (OSS-Me(O] and OOS-trimethyl phosphorothioate (OOS-Me(O] the methyl transferred to the sulphydryl of glutathione is that attached to phosphorus via an oxygen atom. Fractionation of liver cytosol has shown that although the bulk activity is due to the three isozymes (1-1; 3-4; 1.2), OSS-Me(O) is a general substrate for glutathione S-transferases. The specific activity is low compared with the substrates 1-chloro-2,4-dinitrobenzene and 1,2-dichloro-4-nitrobenzene.

Formation of microsomal cytochrome P-450 complexes studied by the NMR relaxation of water.

Cytochrome P-450 in microsomes from liver of phenobarbital treated and control rats has been studied by light absorption and by magnetic resonance methods (EPR and NMR). The nuclear relaxation rate of water protons was measured for microsomal suspensions in the presence of various reactants of Type I and II. The change of relaxation rates correlates well with the spin state conversion of the heme iron. No competition between eventual inner-sphere water molecules and the reactants seems to occur. The temperature dependence of the low spin to high spin equilibrium was studied by light absorption and was accounted for in the temperature variation of the molar relaxation rates of the two spin states.

Surface potential effects on metal ion binding to phosphatidylcholine membranes 31P NMR study of lanthanide and calcium ion binding to egg-yolk lecithin vesicles.

31P NMR of phosphatidylcholine (lecithin) from egg-yolk in sonicated vesicles has been measured in the presence of various ions. Addition of Ln3+ or Ca2+ shifted the 31P resonance of the phosphate groups of the outer surface of the vesicles. These shifts were measured at varied lanthanide or Ca2+ concentration at different ionic strengths obtained by addition of NaCl. The shifts induced by Tb3+ and Ca2+ have been analyzed using the theory of the diffuse double layer. Corrections were introduced for the effect of the ionic strength on the activities of the ions. The binding efficiency is shown to be controlled by the electrostatic potential produced by the bound cations at the membrane surface. This potential is slightly modified due to weak chloride binding. Binding constants have been derived.

Evidence for two catalytically different binding sites of liver microsomal cytochrome P-450: importance for species and sex differences in oxidation pattern of lidocaine.

When the local anaesthetic drug lidocaine is added to liver microsomes biphasic type I spectral change titration curves can be observed. A high-affinity and a low-affinity phase is observed. In the present study we have found that microsomes from female rats have a dominant high-affinity phase, which can hardly be observed within microsomes from female guinea pigs. Male rats showed an intermediate phase. On incubation of lidocaine at concentrations of 1 micron or less with female rat liver microsomes a larger fraction of the drug was aromatically hydroxylated than deethylated. The opposite was true for guinea pig liver microsomes, and microsomes from male rats were intermediate. The ratio between the formation of deethylated and hydroxylated metabolites increased with the lidocaine concentration and at a lidocaine concentration of 10(-4)M deethylation was the dominant oxidation type in all microsomes. The data suggest that the two spectral phases represent two binding sites of cytochrome P-450 each having a certain "catalytic specificity" - the high affinity catalyzing aromatic hydroxylation and the "low-affinity site" deethylation. This hypothesis is further supported by the observed differential effects of pH and MgCl2 concentration on the two types of oxidation.