Pancreatic ribonuclease-poly G complexes: complete inhibition of poly U hydrolysis

Pancreatic ribonuclease forms large complexes with poly G in 0.1 M acetate buffer solutions (pH 5.4). These are largest when the ratio, of ribonuclease to poly G concentration, is slightly less than 2. Under the same conditions lysozyme forms still larger complexes with poly U, and these are largest when the ratio, of lysozyme to poly U concentration, is about 2.5. The ribonuclease in ribonuclease-poly G complexes digests poly U. Free ribonuclease digests the poly U in lysozyme-poly U complexes. However, when the poly G concentration is about an order of magnitude greater than that required to bind all the ribonuclease, lysozyme-bound poly U is not hydrolyzed.     

Dynamics of the Interactions of Basic Proteins with Polyribonucleotides

Pancreatic ribonuclease was irradiated in the dried state with electrons and then added to acetate buffer solutions that contained different concentrations of polyribonucleotides. Qualitatively similar results were obtained by adding a combination of unirradiated ribonuclease and lysozyme to such solutions. Such solutions scatter light strongly, and the intensity of the scattered light changes with time after mixing. The angular distribution of the scattered light was obtained as a function of time and compared with the rates at which hydrolysis products were formed. The turbidity of the solutions increases rapidly with time at the lower polyribonucleotide concentrations, and seems to result from a complex between inactive ribonuclease, or lysozyme, and oligonucleotides that appear during enzymic hydrolysis of the polynucleotides. The dissymmetry of the scattered light is approximately 5, indicating that the scattering centers are, if spherical, about 1500 A in diameter. The turbidities are remarkably high when one considers the low concentrations of protein and nucleic acid materials that are used.     

Monosize poly(glycidyl methacrylate) beads for dye-affinity purification of lysozyme

Cibacron Blue F3GA was covalently attached onto monosize poly(glycidyl methacrylate) [poly(GMA)] beads for purification of lysozyme from chicken egg white. Monosize poly(GMA) beads, 1.6 microm in diameter, were produced by a dispersion polymerization technique. The content of epoxy groups on the surface of the poly(GMA) sample determined by the HCl-pyridine method (3.8 mmol/g). Cibacron Blue F3GA loading was 1.73 mmol/g. The monosize beads were characterized by elemental analysis, FTIR and SEM. Adsorption studies were performed under different conditions in a batch system (i.e., medium pH, protein concentration, temperature and ionic strength). Maximum lysozyme adsorption amount of poly(GMA) and poly(GMA)-Cibacron Blue F3GA beads were 1.6 and 591.7 mg/g, respectively. The applicability of two kinetic models including pseudo-first order and pseudo-second order model was estimated on the basis of comparative analysis of the corresponding rate parameters, equilibrium adsorption capacity and correlation coefficients. Results suggest that chemisorption processes could be the rate-limiting step in the adsorption process. It was observed that after 10 adsorption-elution cycle, poly(GMA)-Cibacron Blue F3GA beads can be used without significant loss in lysozyme adsorption capacity. Purification of lysozyme from egg-white was also investigated. Purification of lysozyme was monitored by determining the lysozyme activity using Micrococcus lysodeikticus as substrate. The purity of the eluted lysozyme was analyzed by SDS-PAGE and found to be 88% with recovery about 79%. The specific activity of the eluted lysozyme was high as 43,600 U/mg.     

Study of poly(C) and poly(I)-poly(C) complexes with model phospholipid membranes by infrared spectroscopy

The temperature dependence of poly(C) is shown by the infrared spectroscopy to be different for the free polynucleotide and for the polynucleotide in complexes with membranes. The intensity of stretching vibrations of C = 0 bond of poly(C) in the complex appears to be sensitive to the temperature. The intensity of this band is sharply decreased by increasing the temperature. This effect depends upon concentration of Mg2+-cations. Adsorption of poly(I)-poly(C) on the surface of vesicles from phosphatidylcholine results in the increase of the double helix.     

Surfactant-induced refolding of lysozyme

The surfactant-lysozyme interaction was investigated by circular dichroism, fluorescence, UV, dynamic light scattering, surface tension, turbidity measurements and lysozyme activity assay. A new way of refolding of lysozyme was found. It was shown that the lysozyme unfolded by anionic surfactants could be renatured by adding cationic surfactants. That is, lysozyme formed precipitate with anionic surfactants, the precipitates could be dissolved by adding a cationic surfactant solution, and then the lysozyme was refolded to its native state spontaneously. Different couples of anionic surfactants and cationic surfactants including C10SO3/C10NE, C12SO3/C10NE, C10SO3/C12NE, C10SO3/C12NB, C10SO4/C10NE and C12SO4/C10NE (C(n)SO3, C(n)SO4, C(n)NE and C(n)NB represent sodium alkyl sulfonate/sulfate, alkyl triethyl/butyl ammonium bromide respectively) were investigated, all of them gave similar results. The results were explained in terms of the differences between the interaction of anionic-cationic surfactants and that of surfactant-lysozyme. It was thought that the formation of mixed micelles of anionic-cationic surfactants is a more favorable process than that of lysozyme-surfactant complexes, which induces the dissociation of lysozyme-surfactant complexes when cationic surfactants were added.     

Interaction of trivaline with single-stranded polyribonucleotides]

Binding of tripeptide H-Val3-(NH)2-Dns (TVP) to polyribonucleotides was studied by fluorescence methods, circular and flow linear dichroism, equilibrium dialysis and electron microscopy. It was found that TVP binds to poly(U) in monomer, dimer and tetramer forms with binding constants of about 10(3), 40, 18.10(4) M, respectively. The cooperativity parameter for peptide dimer binding is 2000. The peptide forms tetramer complexes with poly(A), poly(C), poly(G) also. The formation of a complex between the peptide tetramer and nucleic acid is accompanied by a significant increase in the fluorescence intensity. The cooperative binding of TVP dimers to poly(U), poly(A), poly(C) is accompanied by a dramatic decrease in the flexibility of polynucleotide chains. However, it has a small effect (if any) on the flexibility of the poly(G) chain. The observed similarity of thermodynamic, optical and hydrodynamic++ properties of TVP complexes with single-stranded and double-stranded nucleic acids may reflect a similarity in the geometries of peptide complexes with nucleic acids. Electron microscopy studies show that peptide binding to poly(U) and dsDNA leads to compactization of the nucleic acids caused by interaction between the peptide tetramers bound to a nucleic acid. At the first stage of the compactization process the well-organized rod-like particles are formed, each consisting of one or more single-stranded polynucleotide fibers. Increasing the peptide concentration stimulates a side-by-side association and folding of the rods with the formation of macromolecular "leech-like" structures with the thickness of 20-50 nm.     

Formation of ion-conducting channels by the membrane attack complex proteins of complement.

The effects of sequential additions of purified human complement proteins C5b-6, C7, C8, and C9 to assemble the C5b-9 membrane attack complex (MAC) of complement on electrical properties of planar lipid bilayers have been analyzed. The high resistance state of such membranes was impaired after assembly of large numbers of C5b-8 complexes as indicated by the appearance of rapidly fluctuating membrane currents. The C5b-8 induced conductance was voltage dependent and rectifying at higher voltages. Addition of C9 to membranes with very few C5b-8 complexes caused appearance of few discrete single channels of low conductance (5-25 pS) but after some time very large (greater than 0.5 nS) jumps in conductance could be monitored. This high macroscopic conductance state was dominated by 125-pS channels having a lifetime of approximately 1 s. The high conductance state was not stable and declined again after a period of 1-3 h. Incorporation of MAC extracted from complement-lysed erythrocytes into liposomes and subsequent transformation of such complexes into planar bilayers via an intermediate monolayer state resulted in channels with characteristics similar to the ones produced by sequential assembly of C5b-9. Comparison of the high-conductance C5b-9 channel characteristics (lifetime, ion preference, ionic-strength dependence) with those produced by poly(C9) (the circular or tubular aggregation product of C9) as published by Young, J.D.-E., Z.A. Cohn, and E.R. Podack. (1986. Science [Wash. DC]. 233:184-190.) indicates that the two are significantly different.     

Lysozyme purification with dye-affinity beads under magnetic field

Magnetic poly(2-hydroxyethyl methacrylate) mPHEMA beads carrying Cibacron Blue F3GA were prepared by suspension polymerization of HEMA in the presence of Fe3O4 nano-powder. Average size of spherical beads was 80-120 microm. The beads had a specific surface area of 56.0m(2)/g. The characteristic functional groups of dye-attached mPHEMA beads were analyzed by Fourier transform infrared spectrometer (FTIR) and Raman spectrometer. mPHEMA with a swelling ratio of 68% and carrying 28.5 micromol CibacronBlueF3GA/g were used for the purification of lysozyme. Adsorption studies were performed under different conditions in a magnetically stabilized fluidized bed (i.e., pH, protein concentration, flow-rate, temperature, and ionic strength). Lysozyme adsorption capacity of mPHEMA and mPHEMA/Cibacron Blue F3GA beads were 0.8 mg/g and 342 mg/g, respectively. It was observed that after 20 adsorption-desorption cycle, mPHEMA beads can be used without significant loss in lysozyme adsorption capacity. Purification of lysozyme from egg white was also investigated. Purification of lysozyme was monitored by determining the lysozyme activity using Micrococcus lysodeikticus as substrate. The purity of the desorbed lysozyme was about 87.4% with recovery about 79.6%. The specific activity of the desorbed lysozyme was high as 41.586 U/mg.     

Polynucleotides. XLIV. Synthesis and properties of poly (2-azaadenylic acid) and poly(2-azainosinic acid).

Chemically synthesized 2-azaadenosine 5'-diphosphate (n2ADP) and 2-azainosine 5'-diphosphate (n2IDP) were polymerized to yield poly(2-azaadenylic acid), poly(n2A), and poly(2-azainosinic acid), poly(n2I), using Escherichia coli polynucleotide phosphorylase. In neutral solution, poly(n2A) and poly(n2I) had hypochromicities of 32 and 5.5%, respectively. Poly(n2A) formed an ordered structure, which had a melting temperature (Rm) of 20 degrees C at 0.15 M salt concentration. Upon mixing with poly(U), poly(n2A) formed a 1 : 2 complex with Tm of 41 degrees C at 0.15 M salt concentration. Poly(n2A) and poly(n2I) formed three-stranded complexes with poly(I), and poly(A), respectively. Poly(n2A) . 2poly(I), poly(A) . 2poly(n2I), and poly(n2A) . 2poly(n2I) complexes had Tm values of 23, 48, and 31 degrees C at 0.15 M salt concentration, respectively. Poly(n2I) formed a double-stranded complex with poly(C), but its Tm was very low.     

The transition state in the folding-unfolding reaction of four species of three-disulfide variant of hen lysozyme: the role of each disulfide bridge

The effects of lacking a specific disulfide bridge on the transition state in folding were examined in order to explore the folding-unfolding mechanism of lysozyme. Four species of three-disulfide variant of hen lysozyme (3SS-lysozyme) were prepared by replacing two Cys residues with Ala or Ser: C6S/C127A, C30A/C115A, C64A/C80A and C76A/C94A. The recombinant hen lysozyme was studied as the standard reference containing four authentic disulfide bridges and the extra N-terminal Met: the recombinant hen lysozyme containing the extra N-terminal. Folding rates were measured by monitoring the change in fluorescence intensity associated with tri-N-acetyl-d-glucosamine binding to the active site of refolded lysozyme. It was confirmed that the folding rate of the recombinant hen lysozyme containing the extra N-terminal was the same as that of wild-type lysozyme, and that the folding rate was little affected by the presence of tri-N-acetyl-d-glucosamine (triNAG). The folding rate of C64A/C80A was found to be the fastest and almost the same as that of the recombinant hen lysozyme containing the extra N-terminal, and that of C30A/C115A the second, and that of C6S/C127A the third. The folding rate of C76A/C94A was particularly slow. On the other hand, the unfolding rates which were measured in the presence of triNAG showed the dependence on the concentration of triNAG. The intrinsic unfolding rate in the absence of triNAG was determined by extrapolation. Also in the unfolding rate, C76A/C94A was markedly slower than the others. It was found from the analysis of binding constants of triNAG to C64A/C80A during the unfolding process that the active site of C64A/C80A partly unfolds already prior to the unfolding transition. On the basis of these kinetic data, we suggest that C64A/C80A folding transition can occur with leaving the loop region around SS3 (C64-C80) flexible, while cross-linking by SS4 (C76-C94) is important for the promotion of folding, because it is an indispensable constraint on the way towards the folding transition state.     

High-performance amperometric biosensors and biofuel cell based on chitosan-strengthened cast thin films of chemically synthesized catecholamine polymers with glucose oxidase effectively entrapped

Rapid oxidation of dopamine (DA) or L-noradrenaline (NA) by K(3)Fe(CN)(6) yields poly(DA) (PDA(C)) or poly(NA) (PNA(C)) with glucose oxidase (GOx) effectively entrapped, and such an enzyme-entrapped catecholamine polymer is cast on an Au electrode followed by chitosan (CS) strengthening for biosensing and fabrication of a biofuel cell (BFC). The optimized glucose biosensor of CS/PDA(C)-GOx/Au displays an extremely high sensitivity up to 135 μA mM(-1) cm(-2), a very low limit of detection of 0.07 μM, a response time of <3 s, good suppression of interferents, striking thermostability (lifetime of 3 weeks at 60°C and over 2 months at 30°C), and high resistance to urea denaturation. The biosensor also works well in the second generation biosensing mode with p-benzoquinone (BQ) or ferrocene monocarboxylic acid (Fc) as an artificial mediator, with greatly broadened linear detection ranges (2.0 μM-48.0 mM for BQ and 2.0 μM-16.0 mM for Fc) and up to mA cm(-2)-scale glucose-saturated current density. The good permeability of artificial mediators across the enzyme film enables the quantification of the surface concentration of immobilized GOx on the basis of a reported kinetic model, and UV-Vis spectrophotometry is used to measure the enzymatic activity, revealing high enzymatic activity/load at CS/PDA(C)-GOx/Au. A BFC is also successfully fabricated with a bioanode of CS/PDA(C)-GOx/Au in phosphate buffer solution containing 100 mM glucose and 4.0 mM BQ and a carbon cathode in Nafion-membrane-isolated acidic KMnO(4), and its maximum power density of 1.62 mW cm(-2) is superior to those of most BFC hitherto reported.     

Quantifying anisotropic solute transport in protein crystals using 3-D laser scanning confocal microscopy visualization

The diffusion of a solute, fluorescein into lysozyme protein crystals has been studied by confocal laser scanning microscopy (CLSM). Confocal laser scanning microscopy makes it possible to non-invasively obtain high-resolution three-dimensional (3-D) images of spatial distribution of fluorescein in lysozyme crystals at various time steps. Confocal laser scanning microscopy gives the fluorescence intensity profiles across horizontal planes at several depths of the crystal representing the concentration profiles during diffusion into the crystal. These intensity profiles were fitted with an anisotropic model to determine the diffusivity tensor. Effective diffusion coefficients obtained range from 6.2 x 10(-15) to 120 x 10(-15) m2/s depending on the lysozyme crystal morphology. The diffusion process is found to be anisotropic, and the level of anisotropy depends on the crystal morphology. The packing of the protein molecules in the crystal seems to be the major factor that determines the anisotropy.     

Nanoprecipitation versus emulsion-based techniques for the encapsulation of proteins into biodegradable nanoparticles and process-related stability issues

The goal of this study was to investigate the entrapment of 3 different model proteins (tetanus toxoid, lysozyme, and insulin) into poly(D,L-lactic acid) and poly(D,L-lactic-co-glycolic acid) nanoparticles and to address process-related stability issues. For that purpose, a modified nanoprecipitation method as well as 2 emulsion-based encapsulation techniques (ie, a solid-in oil-in water (s/o/w) and a double emulsion (w₁/o/w₂) method) were used. The main modification of nanoprecipitation involved the use of a wide range of miscible organic solvents such as dimethylsulfoxide and ethanol instead of the common acetone and water. The results obtained showed that tetanus toxoid and lysozyme were efficiently incorporated by the double emulsion procedure when ethyl acetate was used as solvent (>80% entrapment efficiency), whereas it was necessary to use methylene chloride to achieve high insulin entrapment efficiencies. The use of the s/o/w method or the formation of a more hydrophobic protein-surfactant ion pair did not improve protein loading. The nanoprecipitation method led to a homogenous population of small nanoparticles (with size ranging from ≈130 to 560 nm) and in some cases also improved experimental drug loadings, especially for lysozyme (entrapment efficiency >90%). With respect to drug content determination, a simple and quick matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) method provided results very close to those obtained by reverse phase-high-performance liquid chromatography. With respect to protein stability, the duration and intensity of sonication were not a concern for tetanus toxoid, which retained more than 95% of its antigenicity after treatment for 1 minute. Only a high methylene chloride:water ratio was shown to slightly decrease toxoid antigenicity. Finally, no more than 3.3% of A21 desamido insulin and only traces of covalent insulin dimer were detected in nanoparticles. In conclusion, both the double emulsion and nanoprecipitation methods allowed efficient protein encapsulation. MALDI-TOF MS allowed accurate drug content determination. The manufacturing processes evaluated did not damage the primary structure of insulin. Published: December 1, 2005     

B----Z DNA conformational changes induced by a family of dinuclear bis(platinum) complexes.

The reactions of bis(platinum) complexes of general formula [(PtClm(NH3)3-m)2(NH2(CH2)nNH2)]2(2-m)+ were studied with poly(dG-dC).poly(dG-dC), poly(dG-m5dC).poly(dG-m5dC) and poly(dG).poly(dC). When m = 0 (Complexes II, n = 2,4) the complexes are saturated 4+ cations capable only of electrostatic interactions with the polynucleotide. Where m = 1 the complexes contain two monodentate platinum coordination spheres with the chloride trans to the diamine bridge (Complexes I, n = 2,4, 1,1/t,t). Complexes I give CD spectra characteristic of a 'Z-like' conformation upon reaction with poly(dG-dC).poly(dG-dC) and poly(dG-m5dC).poly(dG-m5dC) but not poly(dG).poly(dC). The B----Z transition appears independent of interplatinum diamine chain length. As little as 1 bis(platinum) complex per 25-30 base pairs is sufficient to observe the Z-like spectrum. Covalent binding is however not a prerequisite for Z-DNA formation because the polyvalent cations II are also very effective in inducing the B----Z transition in either poly(dG-dC).poly(dG-dC) or poly (dG-m5dC).poly(dG-m5dC). In these cases, the concentrations of II required are significantly lower than analogous monomeric agents such as [Co(NH3)6]3+. The possible biological consequences of the Z-DNA induction by bis(platinum) complexes are discussed.     

Salt-induced conformational transition of poly(d2NH2A-dT) studied by ultraviolet resonance Raman spectroscopy.

The conformational changes of poly(d2NH2A-dT) in aqueous solution, induced by increasing the NaCl concentration from 0.1M to 4M, have been monitored by ultraviolet resonance Raman spectroscopy, in using the 222-, 257- and 281 nm excitation wavelengths. These changes have been interpreted in comparing the polymer spectra to those of the mononucleotide compounds on one hand, and to those of other alternating purine-pyrimidine polymers on the other hand, i.e. poly(dG-dC) and poly(dA-dT) which showed a B to Z transition in going from low- to high salt concentrations. The high salt poly(d2NH2A-dT) spectra do not show any Raman marker line of the Z conformation. The spectroscopic results indicate that most of the ribose puckering goes from C2'-endo/anti to C3'-endo/anti in increasing the salt concentration. In addition the base stacking interactions, to which the resonance Raman effect is very sensitive, are not drastically changed upon salt variations. Thus the high salt structure of poly(d2NH2A-dT) remains a right-handed helix, likely under a dominant A conformation.     

Rodlike complexes of a polyelectrolyte (hyaluronan) and a protein (lysozyme) observed by SANS

We study by small-angle neutron scattering (SANS) the structure of hyaluronan -lysozyme complexes. Hyaluronan (HA) is a polysaccharide of 9 nm intrinsic persistence length that bears one negative charge per disaccharide monomer (M(mol) = 401.3 g/mol); two molecular weights, M(w) = 6000 and 500,000 Da were used. The pH was adjusted at 4.7 and 7.4 so that lysozyme has a global charge of +10 and +8, respectively. The lysozyme concentration was varied from 3 to 40 g/L at constant HA concentration (10 g/L). At low protein concentration, samples are monophasic, and SANS experiments reveal only fluctuations of concentration, although, at high protein concentration, clusters are observed by SANS in the dense phase of the diphasic samples. In between, close to the onset of the phase separation, a distinct original scattering is observed. It is characteristic of a rod-like shape, which could characterize "single" complexes involving one or a few polymer chains. For the large molecular weight (500,000), the rodlike rigid domains extend to much larger length scale than the persistence length of the HA chain alone in solution and the range of the SANS investigation. They can be described as a necklace of proteins attached along a backbone of diameter of one or a few HA chains. For the short chains (M(w) ≈ 6000), the rod length of the complexes is close to the chain contour length (～ 15 nm).     

Mechanism of lysozyme catalysis: role of ground-state strain in subsite D in hen egg-white and human lysozymes.

The association constants for the binding of various saccharides to hen egg-white lysozyme and human lysozyme have been measured by fluorescence titration. Among these are the oligosaccharides GlcNAc-beta(1 leads to 4)-MurNAc-beta(1 leads to 4)-GlcNAc-beta(1 leads to 4)-GlcNAc, GlcNAc-beta(1 leads to 4)-MurNAc-beta(1 leads to 4)-GlcNAc-beta(1 leads to 4)-N-acetyl-D-xylosamine, and GlcNAc-beta(1 leads to 4-GlcNAc-beta(1 leads to 4)-MurNAc, prepared here for the first time. The binding constants for saccharides which must have N-acetylmuramic acid, N-acetyl-D-glucosamine, or N-acetyl-D-xylosamine bound in subsite D indicate that there is no strain involved in the binding of N-acetyl-D-glycosamine in this site, and that the lactyl group of N-acetylmuramic acid (rather than the hydroxymethyl group) is responsible for the apparent strain previously reported for binding at this subsite. For hen egg-white lysozyme, the dependence of saccharide binding on pH or on a saturating concentration of Gd(III) suggests that the conformation of several of the complexes are different from one another and from that proposed for a productive complex. This is supported by fluorescence difference spectra of the various hen egg-white lysozyme-saccharide complexes. Human lysozyme binds most saccharides studied more weakly than the hen egg-white enzyme, but binds GlcNAc-beta(1 leads to 4)-MurNAc-beta(1leads to 4)-GlcNAc-beta(1 leads to 4)-MurNAc more strongly. It is suggested that subsite C of the human enzyme is "looser" than the equivalent site in the hen egg enzyme, so that the rearrangement of a saccharide in this subsite in response to introduction of an N-acetylmuramic acid residue into subsite D destabilizes the saccharide complexes of human lysozyme less than it does the corresponding hen egg-white lysozyme complexes. This difference and the differences in the fluorescence difference spectra of hen egg-white lysozyme and human lysozyme are ascribed mainly to the replacement of Trp-62 in hen egg-white lysozyme by Tyr-63 in the human enzyme. The implications of our findings for the assumption of superposition and additivity of energies of binding in individual subsites, and for the estimation of the role of strain in lysozyme catalysis, are discussed.     

Thermoprecipitation of lysozyme from egg white using copolymers of N-isopropylacrylamide and acidic monomers

Thermoprecipitation of lysozyme from egg white was demonstrated using copolymers of N-isopropylacrylamide with acrylic acid, methacrylic acid, 2-acryloylamido-2-methylpropane-sulfonic acid and itaconic acid, respectively. Polymers synthesized using molar feed ratio of N-isopropylacrylamide:acidic monomers of 98:2 exhibited lower critical solution temperatures in the range of 33--35 degrees C. These polymers exhibited electrostatic interactions with lysozyme and inhibited its bacteriolytic activity. The concentration of acidic groups required to attain 50% relative inhibition of lysozyme by the polymers, was 10(4)--10(5) times lower than that required for the corresponding monomers. This was attributed to the multimeric nature of polymer-lysozyme binding. More than 90% lysozyme activity was recovered from egg white. Polymers exhibited reusability up to at least 16 cycles with retention of >85% recovery of specific activity from aqueous solution. In contrast, copolymer comprising natural inhibitor of lysozyme i.e. poly (N-isopropylacrylamide-co-O-acryloyl N-acetylglucosamine) lost 50% recovery of specific activity. Thermoprecipitation using these copolymers, which enables very high recovery of lysozyme from egg white, would be advantageous over pH sensitive polymers, which generally exhibit lower recovery.     

Degradation of fully water-soluble, partially N-acetylated chitosans with lysozyme

Chitosans, prepared by homogeneous N-deacetylation of chitin, with degrees of N-acetylation ranging from 4 to 60% (F_A = 0·04 to 0·60) exhibiting full water solubility and known random distribution of acetyl groups, were degraded with lysozyme. Initial degradation rates (r) were determined from plots of the viscosity decrease (Δ1/[η]) against time of degradation. The time course of degradation of chitosans with lysozyme were non-linear, while the time course of degradation of chitosans with an oxidative-reductive depolymerization reaction (using H₂O₂) showed the expected linear relationship for a first-order, random depolymerization reaction, independent of the chemical composition of the chitosan.

The effect of lysozyme concentration and substrate concentration on the initial degradation rates were determined, showing that this lysozyme-chitosan system obeys Michaelis-Menten kinetics.

The initial degradation rates of chitosan with lysozyme increased strongly with increasing fraction of acetylated units (F_A). From a Michaelis-Menten analysis of the degradation data that assumes different catalytic activities of lysozyme for the different hexameric substrates in the polysaccharide chain, it is concluded that the hexameric substrates that contain three-four or more acetylated units contribute mostly to the initial degradation rate when lysozyme degrades partially N-acetylated chitosans.

A chitosan with a very low fraction of acetylated units (F_A = 0·010) was studied as an enzyme inhibitor. Initial degradation rates of chitosan (with different F_A values) decreased as the inhibitor concentration increased, while the relative rates stayed constant, indicating that the ratio between initial reaction rates for productive sites (hexamers containing three-four or more N-acetylated units) are unaffected by non-productive sites, as deduced from the theory of competing substrates.