Enzymic and immunochemical properties of lysozyme. X. Conformation, enzymic activity and immunochemistry of lysozyme reduced at two carboxyl groups.

Reduction of lysozyme by diborane, followed by air oxidation of the reduced disulfides and chromatography on CM-cellulose, yielded a homogeneous derivative. In the derivative, the carboxyl groups of aspartic acid 119 and the end-chain leucine residue were reduced to their corresponding alcohols. Correct re-forming of the disulfide bonds was demonstrated by peptide mapping of the tryptic hydrolysates of the derivative and lysozyme without breaking the disulfide bonds, followed by identification of the disulfide-containing peptides. Correct disulfide pairing in the two-disulfide peptide in the tryptic hydrolysate was established from its immunochemical behavior. Preparations of the two-disulfide fragment from lysozyme and derivative had equal inhibitory activities (26 or 32%) of the reaction of lysozyme with two homologous antisera. In ORD measurements, lysozyme and the derivative had equal rotatory powers at neutral pH. However, the bo value for the derivative decreased by about 10%. Below pH 6.4 and above pH 8.0, the derivative was less rotatory than native lysozyme. In CD measurements at neutral pH, the negative ellipticity bands at 220 and 208 nm showed little or no decrease in the derivative relative to the native protein. Although conformational differences between the derivative and its parent protein were almost undetectable by ORD and CD measurements, they were readily detected by chemical monitoring of the conformation. In the derivative, both accessibility to tryptic hydrolysis and reducibility of the disulfide bonds increased markedly. The enzymic activity of the derivative was decreased but retained the same pH optimum. With antisera to lysozyme or antisera to the derivative, lysozyme and its derivative possessed equal antigenic reactivities. The immunochemical findings further confirm the correct refolding of the disulfides. Also, they indicate that aspartic acid 119 and the C-terminal leucine residue are not part of an antigenic reactive region in lysozyme.     

Enzymic and immunochemical properties of lysozyme. IX. Conformation and immunochemistry of derivatives succinylated at certain lysine residues.

Succinylation of lysozyme in the presence of 7 molar excess of [1,4-14C2]-succinic anhydride gave a reaction product which showed at least six components by disc electrophoresis. Chromatography on CM-cellulose enabled the isolation of six homogeneous derivatives. The derivatives were succinylated at the following locations: derivative I, lysines-1 (alpha- and epsilon-NH2), -13, -97 and -116 and the OH group at position 43 (or 36 or 40); derivative II, lysines-1 (alpha- and epsilon-NH2), -13, -96, -116; derivative III, lysines-1 (alpha-and epsilon-NH2), -13, -97, -116; derivative IV, lysines-1 (alpha-NH2), -33, -96 and -116; derivative V, lysines-1 (alpha-NH2), -33 and -96; derivative VI, lysines-33 and -116. Conformational changes were detectable in derivative I by ORD and CD measurements and by accessibility of the disulfide bonds to reduction. On the other hand, the other five succinyl derivatives showed no conformational changes by ORD and CD measurements. However, their disulfide bonds were slightly more accessible to reduction than lysozyme, with the increase being somewhat higher in derivatives I, II and III. Enzymic activity measurements showed that only derivative VI possessed some (10%) enzymic activity. Immunochemical studies with antisera to lysozyme showed that the reactivity of each of the derivatives was lower than the homologous reaction. Correlation of the extent of decrease in immunochemical reaction with the locations of modification and with the results of conformational analysis, led to the conclusion that lysines 33, 96 and 116 are part of antigenic reactive regions in lysozyme. The modification results are also discussed in relation to the three-dimensional structure of lysozyme in solution.     

Enzymic and immunochemical properties of lysozyme. XI. Conformation and immunochemistry of the two-disulfide peptide and the tryptophan and lysine residues in its antigenic reactivity.

The previously described peptide 62-68 (Cys 64-Cys 80) 74-96 (Cys 76-Cys 94) (Atassi, M.Z., Suliman, A.M. and Habeeb, A.F.S.A. (1975) Biochim. Biophys. Acta 405, 452-463), which accounted for about one-third of the total antigenic reactivity of native lysozyme, was isolated here with lysine 97 attached to it. The peptide was subjected to specific modification reactions in order to determine some of the residues which formed part of its antigenic reactive site. ORD measurements showed that the peptide was greatly unfolded in solution relative to its expected mode of folding within the intact lysozyme molecule. Modification of the two tryptophan residues in the peptide by reaction with 2,3-dioxo-5-indolinesulfonic acid provided a derivative which possessed similar conformational parameters to those of the unmodified peptide. However, the derivative retained only about half the immunochemical reactivity of the peptide. Succinylation of the amino groups afforded a derivative whose conformational parameters were identical to those of the unmodified peptide but in which half of the immunochemical reactivity was lost. Modification of the two tryptophan residues followed by succinylation of the amino groups resulted in almost complete loss of the antigenic reactivity, and the loss was not due to conformational differences. The antigenic reactivity of the peptide was also destroyed on removal of tryptophans 62 and 63, of sequence 84-93 from the loop 74-79 and of sequence 74-75 by chymotryptic digestion. From these and previous results it was concluded that the antigenic reactive site in this part of the lysozyme molecule incorporates one or both of tryptophans 62 and 63 as well as one or both lysines 96 and 97. The two disulfides 64-80 and 76-94 bring these two parts of the lysozyme molecule into a single reactive site. The intactness of the disulfides is essential for maintenance and reactivity of the site.     

Phage T4 lysozyme. Physical properties and reversible unfolding.

Phage T4 lysozyme has been used extensively in studies of the genetic code. However, little work has been done on the characterization of the purified enzyme. Therefore, we determined the spectral properties of native T4 lysozyme and used these properties to follow the unfolding transition. The ultraviolet absorption spectrum and solvent perturbation difference spectrum indicate that the aromatic amino acids are extensively exposed to solvent. The CD and ORD spectra are characteristic of a high fraction of helix. Guanidine hydrochloride denaturation results show that over a T4 lysozyme concentration range of 0.07-1 g/l the c-m equals 2.7 M guanidine hydrochloride at pH 5 and that the transition is 100% reversible as judged by enzymatic assay and four different spectrophotometric criteria: CD at 295 nm, CD at 223 nm, fluorescence intensity at 350 nm and wavelength of maximum fluorescence. Guanidine hydrochloride denaturation at pH 2.5 was followed using fluorescence emission and has a c-m equals 1.7 M guanidine hydrochloride, indicating a strong pH dependence of chemical unfolding. Reversible thermal denaturation conditions were located at acid pH, 0.2 M NaCl, 10-4 M dithiothreitol and 10-6 M T4 lysozyme. The CD signal at 223 nm was used to measure the unfolding. Thermodynamic analysis of the thermal data showed an increase in T-m, increment H-unf and increment S-unf with increasing pH.     

Total optical activity of agarose: relation to observable transitions in the vacuum ultraviolet

The changes in optical activity that accompany and characterize the coil-helix and helix-coil transitions of agarose in aqueous solutions and gels have been investigated by combined quantitative analysis of data from vacuum ultraviolet circular dichroism (VUCD) and optical rotary dispersion (ORD). VUCD of agarose in the high-temperature coil state shows a single accessible Gaussian band centered at ～183 nm. In the helix state this band is blue-shifted by ～9 nm, and the intensity is increased by a factor of ～2.6. Spectra at intermediate temperatures can be fitted to within experimental error by linear combination of coil and helix spectra, the relative proportions required providing an index of the extent of conformational ordering. ORD spectra throughout the conformational transition have a common form and differ only in absolute magnitude. The temperature course of conformational ordering derived from ORD intensity is in close agreement with the values obtained from VUCD. In both the coil and helix states the accessible VUCD band is positive, while the overall ORD is negative, indicating strong negative CD activity at lower wavelength. The ORD contribution corresponding to the positive VUCD band was calculated by Kronig–Kramers transform, and it was subtracted from the total ORD to give the residual ORD from all other optically active transitions of the molecule. In both the coil and helix states, this residual ORD could be fitted to within experimental error by a single Gaussian CD band at ～149 nm. A negative band at this wavelength has been reported previously for agarose films, but the observed intensity, relative to that of the lower energy positive band, is substantially smaller than the fitted value under hydrated conditions. In both the coil and helix states the total optical activity of agarose, characterized by observed ORD spectra, can be matched to within experimental error by Kronig-Kramers transform of the 149-nm negative band and the smaller positive band at higher wavelength, with no necessary involvement of deeper-lying transitions. The significance of this conclusion for fundamental understanding of carbohydrate optical activity is discussed.     

Far-ultraviolet optical activity of saccharide derivatives. II. Dextran,amylose, and mycodextran acetes; dextran and amylose xanthates

The ultraviolet ORD and CD spectra of amylose, dextran, and mycodextran acetates and some of thier oligomers were recorded in trifluoroethanol solution in the 300–185nm wavelength range. Similarly, the spectra of amylose and dextran xanthates in water solution were obtained in the 400–200 nm range. In the amylose acetate series, the monomer and dimer both show a normal acetyl n → π* transition in CD, while the trimer and the polymer both exhibit an additional, shorter wavelength peak. The latter is presumed to arise from a helical conformation of the amylose chain. This interpretation is substantiated by a helix–coil type transition of the CD spectra of amylose triacetate at elevated temperatures and a reversion of the anomalous CD to the normal CD upon partial deacetylation. By contrast, neither dextran acetates nor mycodextran acetate exhibit any conformational effects. The CD of dextran acetates is quite sensitive to β-1,6 and branch linkages. The ORD and CD of amylose xanthate are complex, suggesting the presence of organized structure in solution. The dextran xanthate shows only a simple ORD spectrum and no observable CD.     

Conformational studies on a modified poly-L-tryptophan: Circular dichroism and optical rotatory dispersion of poly-2-(2-nitrophenylsulfenyl)-L-tryptophan and of random copolymers of L-tryptophan and 2-(2-nitrophenylsulfenyl)-L-tryptophan

Optical rotatory dispersion (ORD) and circular dichroism (CD) measurements were carried out on a block copolymer, (γ-ethyl DL -glutamate)₁₆₀ (L -Trp)₃₂, in which the tryptophan sequence has been modified to various extents by using 2-nitrophenylsulfenyl chloride. The CD spectrum of the completely modified copolymer exhibits bands in some of the regions of maximum absorption of the sidechain chromophores. In the peptide absorption region the spectrum is similar to that reported in the literature for polypeptides in the α-helical conformation. When the extent of modification of the tryptophan sequence is progressively reduced, there is a gradual change in the ORD spectra of the copolymers. On the basis of these data the assumption was made that no conformational change occurs on proceeding from the pure unmodified tryptophan sequence to the completely modified sequence. The results are discussed in connection with the study of possible conformational effects arising from selective chemical modification of tryptophan residues in proteins.     

Studies on pig serum lipoproteins. V. Optical properties of low density lipoproteins.

The circular dichroism (CD), optical rotatory dispersion (ORD), and fluorescence emission spectra of two subfractions of pig serum low density lipoproteins (LDL1 and LDL2) were compared. The contribution of the carbohydrate moiety to the CD and ORD spectra was estimated on the basis of data obtained from isolated glycopeptides and the constituent monosaccharides. The carbohydrate moiety had no effect on the conformation of the protein moieties of LDL1 and LDL2 (apoLDL1 and apoLDL2). However, the intensities of the observed extrema in the CD and ORD spectra of the glycopeptides were greater than those expected from the monosaccharide composition. This suggests the existence of secondary structure in the carbohydrate moiety. In contrast to the carbohydrate moiety, the contribution of the lipid moiety to the CD and ORD spectra could not be neglected. When the effect of the lipid moiety was subtrated from the CD and ORD spectra, the spectra due to apoLDL1 and apoLDL2 were quite similar. Delipidation in the presence of sodium dodecyl sulfate (SDS) induced an increase in the content of disordered structure and alpha-helix accompanied by a decrease in the beta-structure. In the presence of SDS, marked quenching occurred in the fluorescence emission spectra with a blue shift of the maximum emission wavelength from 332 to 326 nm. ApoLDL1 and apoLDL2 showed quite similar SDS-induced conformational transitions. The secondary structures of apoLDL1 and apoLDL2 in the native lipoproteins were stable to changes of pH and temperature. However, this stability was lost in the presence of SDS. These results suggest the importance of the lipid moiety in maintaining the native secondary structures of LDL1 and LDL2. From the overall similarity of the optical properties of apoLDL1 and apoLDL2, we conclude that the secondary structures of apoLDL1 and apoLDL2 are identical.     

Influence of phosphate ligands in abolishing the conformational difference between ribonuclease A and its acid-denatured derivative.

The initial structural alteration of RNAase A due to acid denaturation (0.5 N HCl, 30 degrees C) that accompanies deamidation (without altering enzymic activity) has been dectected by spectrophotometric titration, fluorescence and ORD/CD measurements. It is shown that acid treated RNAase A has an altered conformation at neutral pH, 25 degrees C. This is characterized by the increased accessibility of buried tyrosine residue(s) towards the solvent. The most altered conformation of RNAase A is found in the 10 h acid-treated derivative. This has about 1.5 additional exposed tyrosine residues and a lesser amount of secondary structure than RNAase A. All three methods (titration, fluorescence and CD) established that the structural transition of RNAase A is biphasic. The first phase occurs within 1 h and the resulting subtle conformational change is constant up to 7 h. Following this, after the release of 0.55 mol of ammonia, the major conformational change begins. The altered conformation of the acid-denatured RNAase A could be reversed completely to the native state through a conformational change induced by substrate analogs like 2'- or 3'-CMP. Thus the monodeamidated derivative isolated from the acid-denatured RNAase A by phosphate is very similar to RNAase A in over-all conformation. The results suggest the possibility of flexibility in the RNAase A molecule that does not affect its catalytic activity, as probed through the tyrosine residues.     

Partially unfolded equilibrium state of hen lysozyme studied by circular dichroism spectroscopy

Equilibrium unfolding of hen egg white lysozyme as a function of GdnCl concentration at pH 0.9 was studied over a temperature range 268.2-303.2 K by means of CD spectroscopy. As monitored by far- and near-UV CD at 222 and 289 nm, the lack of coincidence between two unfolding transition curves was observed, which suggests the existence of a third conformational species in addition to native and unfolded states. The three-state model, in which a stable intermediate is populated, was employed to estimate the thermodynamic parameters for the GdnCl-induced unfolding. It was found that the transition from the native to intermediate states proceeds with significant changes in enthalpy and entropy due to an extremely cooperative process, while the transition from the intermediate to unfolded states shows a low cooperativity with small enthalpy and entropy changes. These results indicate that the highest energy barrier for the GdnCl-induced unfolding of hen lysozyme is located in the process from the native state to the intermediate state, and this process is largely responsible for the cooperativity of protein unfolding.     

Interaction of lysozyme with dyes. II. Binding of bromophenol blue

The binding of lysozyme with bromophenol blue (BPB) at various dye concentrations and pH was carried out at 25 degrees C by equilibrium dialysis, ultraviolet (UV) difference and circular dichroism (CD) spectral techniques. Binding isotherms at pH 5.0 show non-cooperative binding at low dye concentrations, which change over to cooperative binding at higher concentrations indicating biphasic nature. However, binding isotherms at pH 7.0 and 9.0 show cooperative binding only, at all concentrations of the dye. The number of available binding sites decreases with the increase of pH. Gibbs free energy change, calculated on the basis of Wyman's binding potential concept, decreases with the increase of pH. Binding isotherms at pH 5.0 obtained at a lower temperature of 8 degrees C, also indicate the biphasic nature similar to those observed at 25 degrees C, but with a slight decreased strength of binding. The UV difference spectra of the complex do not show any distinct peaks in the 285 to 297 nm region eliminating any possible interaction of BPB with tryptophan and tyrosine residues of the lysozyme molecule. The CD spectra of lysozyme-BPB complex show a decrease in ellipticities with reference to native lysozyme in the near UV and far UV regions. This indicates that the lysozyme-BPB complex has a lower helical content probably due to the conformational changes induced into the native enzyme. The appearance of new positive peaks at 315 nm in the near UV region and at 592 nm in the visible region of the CD spectra may be due to the induced asymmetry into the BPB molecule as a result of its binding to a cationic residue (probably a lysine residue) of lysozyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Optical Rotatory Dispersion and Circular Dichroism of Amino Acid Hydantoins

Optical rotatory dispersion (ORD) and circular dichroism (CD) of 17 amino acid hydantoins were measured between 190 and 600 nm. Most of hydantoins exhibited the negative Cotton effect which showed the trough between 238 and 245 nm. The negative trough of CD was also observed between 212 and 236 nm. The Cotton effect of hydantoins was attributable to n→π^* transition of carbonyl group at C-4 of hydantoin ring.     

Chiroptic recognition of potassium ion

The high specificity in the recognition and specific binding of potassium ion by the depsipeptide valinomycin (VM) is exploited for its recognition and quantitation using both circular dichroism (CD) and optical rotation dispersion (ORD). The specific rotation of VM is comparably small (2.34 deg ml g(-1) cm(-1)), so that an 8 microM (= 8.89 mg ml(-1)) solution of VM in 95% ethanol rotates polarized light of Lambda = 426 nm passing a 2 cm cuvette by 0.076 degrees only. It is shown, however, that VM undergoes large changes in both ORD and CD on binding to potassium ion. VM, potassium ion and the anionic dye merocyanine 540 form a ternary complex (VM/K/MC) which displays an induced CD with a positive maximum at 488 nm and a negative maximum at 470 nm. The ternary complex also displays fluorescence that is weaker by about 30% when compared to that of the dye alone. The induced CD of the ternary complex is interpreted in terms of the large conformational change which VM is known to undergo on binding potassium ion, thereby forming the prerequisite for a van der Waals interaction between its outwardly directed lipophilic domains and the lipophilic domains of the anionic dye. The method is likely to be applicable to the fluorescent detection of all kinds of ions for which chiral receptors are known, e.g. in studies on the role of ions in biological systems including ion channels.     

Effect of counterions on the conformation of hyaluronic acid.

To study the effect of metal ions on the conformation of hyaluronic acid, circular dichroism (CD) and optical rotatory dispersion (ORD), along with viscosity measurements of the Na, Li, Ca, and Mg salts of the polymer, were carried out. With divalent cations, the results show a decrease in CD minima at 210 nm and an increase in ORD troughs at 220 nm, as compared to monovalent ions. To account for this behavior, the ORD in the visible range corresponding to the observed CD bands was directly calculated from the Moscowitz equation using Kronig-Kramer's transform. The background rotation was found to be more levorotatory in bivalent than in monovalent cations. The ORD spectra of various metal hyaluronates differ significantly from each other in the far ultraviolet region, especially at lower pH values. The values of intrinsic viscosities of these hyaluronates, on the other hand, are almost the same in the pH range of 1–3. These results indicate a local conformation variation rather than any appreciable change in the chain conformation of the molecule in the presence of different counterions.     

The precise and entire antigenic structure of native lysozyme.

The exact boundary, residue, conformational and directional definitions of the three antigenic sites of native hen's egg-white lysozyme are described. The results clearly reveal that the three antigenic sites account quantitatively for the total antigenic reactivity of the protein. Thus the entire antigenic structure of lysozyme has now been precisely determined and is briefly discussed here, together with the power of the surface-stimulation synthetic concept.     

Volatile anesthetics cause conformational changes of bacteriorhodopsin in purple membrane

We examined the effects of volatile anesthetics on the structure of the bacteriorhodopsin in the purple membrane by measurements of the absorption spectrum and the visible circular dichroism (CD) spectrum and assay of the retinal composition. As the concentrations of halothane, enflurane and methoxyflurane were increased, the absorption at 560 nm decreased but that at 480 nm increased with an isosbestic point around 510 nm. These anesthetic-induced spectroscopic changes were reversible. The CD spectrum showed the biphasic pattern with a positive and a negative band. As the concentration of halothane was increased from 4 mM to 8mM, the negative band reversibly diminished more drastically than the positive band, and at 8 mM of halothane the positive band shifted to around 480 nm. These results show that halothane disturbed the exciton coupling among bacteriorhodopsin molecules. The retinal isomer composition was analyzed using high performance liquid chromatography. The ratio of 13-cis- to all-trans-retinal was 47:53, 34:66 and 19:81 at control, 7.4 mM and 14.9 mM enflurane, respectively. After elimination of enflurane, the ratio returned to the control value. These findings indicate that volatile anesthetic directly affect a bacteriorhodopsin in the purple membrane and induce conformational changes in it.     

Difference absorption spectra, circular dichroism, and disulfide cleavage of hen and turkey lysozymes in the alkaline pH region.

The difference absorption spectra of hen and turkey lysozymes in the alkaline pH region had three maxima at around 245, 292, and 300 nm and had no isosbestic points. The ratio of the extinction difference at 245 nm to that at 295 nm changed with pH. These spectral features are quite different from those observed when only tyrosyl residues are ionized, and it was impossible to determine precisely the pK values of the tyrosyl residues in lysozyme by spectrophotometric titration. A time-dependent spectral change was observed above about pH 12. This is not due to exposure of a buried tyrosyl residue on alkali denaturation. The disulfide bonds and the peptide bonds in the lysozyme molecule were cleaved by alkali above about pH 11. The intrinsic pK value of Tyr 23 of hen lysozyme was determined to be 10.24 (apparent pK 9.8) at 0.1 ionic strength and 25 degrees C from the CD titration data. Comparison of the CD titration of turkey lysozyme with that of hen lysozyme suggested that Tyr 3 and Tyr 23 in turkey lysozyme have apparent pK values of 11.9 and 9.8, respectively.     

Optical rotary dispersion of some heptaene antibiotics]

Experimental differences in the curves of the optic rotation dispersion (ORD) of cystrans-heptaenic antibiotics were found. The ORD curves of amphotericin B, mycoheptin, levorin components and isolevorin A2, components of criptomycin and candidin were registered. The curves of the ORD which were smooth had been prepared in dimethylsulphoxide in the spectral range at 450 to 600 nm. In the spectral range at 300 to 420 nm the ORD curves appeared to be anomal with a complex Kotton effect, they were prepared in methyl alcohol. The Kotton effect was probably due to asymmetry of the electron membranes of polyenic chromophore induced by the other part of the polyen molecule. This was evident from the fact that the curve of the Kotton effect was situated in the same spectral range as the absorption bond of the polyenic chromophore. The oscillating structure in the absorption spectrum and the curve of the complex Kotton effect were analogous.     

Spectroscopic, immunochemical, and thermodynamic properties of carboxymethyl(Cys6, Cys127)-hen egg white lysozyme

A three-disulfide form of hen egg white lysozyme with Cys⁶ and Cys¹²⁷ blocked by carboxymethyl groups was prepared, purified, and characterized for eventual use in protein folding experiments. Trypsin digestion followed by proline-specific endopeptidase digestion facilitated the unambiguous assignment of the disulfide bond pairings and the modified residues in this derivative. 3SS-lysozyme demonstrated nearly full enzymatic activity at itspH optimum,pH 5.5. The 3SS-lysozyme derivative and unmodified lysozyme were shown to be identical by CD spectroscopy atpH 3.6. Immunochemical binding assays demonstrated that the conformation of lysozyme was perturbed predominantly only locally by breaking and blocking the disulfide bond between Cys⁶ and Cys¹²⁷. Both 3SS-lysozyme and unmodified lysozyme exhibited reversible thermally induced transitions atpH 2.0 but theT _m of 3SS-lysozyme, 18.9°C, was found to be 34° lower than that of native lysozyme under the same conditions. The conformational chemical potential of the denatured form of unmodified lysozyme was determined from the transition curves to be approximately 6.7 kcal/mol higher than that of the denatured form of 3SS-lysozyme, atpH 2.0 and 35°C, if the conformational chemical potential for the folded forms ofboth 3SS-lysozyme and unmodified lysozyme is arbitrarily assumed to be 0.0 kcal/mol. A calculation of the increase in the theoretical loop entropy of denatured 3SS-lysozyme resulting from the cleavage of the Cys⁶-Cys¹²⁷ disulfide bond, however, yielded a value of only 5.4 kcal/mol for the difference in conformational chemical potential. This suggests that, in addition to the entropic component, there is also an enthalpic contribution to the difference in the conformational chemical potential corresponding to approximately 1.3 kcal/mol. Thus, it is concluded that the reduction and blocking of the disulfide bond between Cys⁶ and Cys¹²⁷ destabilizes 3SS-lysozyme relative to unmodified lysozyme predominantly by stabilizing the denatured conformation by increasing its chain entropy.Cornell Biotechnology Army Research Office Predoctoral Fellow, 1986–1989.     

Action of myeloperoxidase-hydrogen peroxide-chloride system on the egg white lysozyme

The enzyme system composed of human neutrophilic myeloperoxidase (H2O2-oxidoreductase, EC 1.11.1.7), H2O2 and Cl-, at pH 4.5 interacts with egg white lysozyme (EC 3.2.1.17) in several stages. In the first stage, occurring at lysozyme to H2O2 molar ratio of 1:1.4-1.8, the lysozyme loses its enzyme activity but does not yield any derivative distinguishable from the native protein on polyacrylamide gel electrophoresis (PAGE). The second stage of oxidation begins at lysozyme to H2O2 molar ratio above 1:5, producing a change in the lysozyme spectrum at 260-290 nm, and yielding protein derivatives with molecular masses equal to multiples of 14.3 kDa, i.e. the lysozyme molecular mass. This implies that an excessive oxidation of lysozyme by the myeloperoxidase-H2O2-Cl- system produces cross-linking of lysozyme molecules to di-, tri-, tetra-, and pentameric structures. At lysozyme to H2O2 molar ratio exceeding 1:12 a water insoluble white product, which consists of a set of lysozyme cross-linked derivatives, is obtained.