Sorbitol prevents the self-aggregation of unfolded lysozyme leading to and up to 13 degrees C stabilisation of the folded form

We present a calorimetric investigation of stabilisation of hen egg-white lysozyme with sorbitol in the pH range 3.8-10.5. Differential scanning calorimetry and steady-state fluorescence were used to determine the denaturation temperatures of lysozyme as a function of sorbitol concentration. The fluorescence data were collected in the presence of 2M urea to lower the melting point of the protein to an observable range of the instrument. The effect of sorbitol on the activation energy of unfolding was investigated by scanrate studies. The effect of sorbitol lysozyme interaction was investigated using isothermal titration calorimetry. The titration experiments were performed with folded as well as unfolded lysozyme to investigate in more detail the nature of the interaction. The data obtained in those experiments show a remarkable stabilisation effect of sorbitol. We observed a 4.0 degrees C increase in the Tm for 1 M sorbitol in the pH range 3.8-8.5 by scanning calorimetry. The effect increases dramatically at pH 9.5 where we observe a 9.5 degrees C stabilisation. An increase in the sorbitol concentration to 2 M stabilises lysozyme by 11.3-13.4 degrees C in the pH range 9.5-10.5. In the absence of urea, no significant effects of sorbitol were observed on the activation energy for unfolding for lysozyme at pH 4.5. This indicates together with the results from the titration experiments that sorbitol may stabilise the folded form of lysozyme by destabilising the unfolded form of lysozyme. At pH values at and above lysozyme's pI (approximately 9.3), the unfolding of the protein is accompanied with a substantial amount of self-aggregation seen in the calorimetry experiments in the ratio of DeltaH(cal)/DeltaH(vH). In the presence of sorbitol, the self-aggregation was counterbalanced by higher sorbitol concentrations. These results strongly suggest a negative influence of sorbitol on the unfolded form of lysozyme and thereby stabilising the native form.     

A study of renaturation of reduced hen egg white lysozyme. Enzymically active intermediates formed during oxidation of the reduced protein.

The material obtained from reduced hen egg white lysozyme after complete air oxidation at pH 8.0 and 37 degrees has yielded, by gel filtration on a Bio-Gel P-30 column, enzymically active species and an enzymically inactive form which eluted sooner than the active species but later than expected for a dimer of lysozyme. Reduced lysozyme also elutes at the same position as this inactive material. Examination of the fragments produced on CNBr cleavage of the inactive form indicates that at least 24% of the population contains incorrect disulfide bonds involving half-cystine residues 6, 30, 115, and 127. Tryptophan fluorescence and the intrinsic viscosity of the inactive form show an enlarged molecular domain with a disordered conformation. The yield of the inactive form increases as the oxidation of reduced lysozyme is accelerated using cupric ion. In the presence of 4 X 10(-5) M cupric ion, reduced lysozyme forms almost quantitatively the inactive form, which is almost completely converted to the native form by sulfhydryl-disulfide interchange catalyzed by thiol groups of either reduced lysozyme or beta-mercaptoethanol. The material trapped by alkylation of the free sulfhydryl groups with [1-14C]iodoacetic acid during the early stage of air oxidation of reduced lysozyme was fractionated by gel filtration to permit separation of the active species from the inactive form. Ion exchange chromatography of the active species yielded completely renatured lysozyme and three major enzymically active radioactive derivatives. Two of these derivatives contained approximately 2 mol of S-carboxymethylcysteine. Isolation and characterization of radioactive tryptic peptides from each of the three active forms, permitted the identification of Cys 6 and Cys 127, Cys 76 and 94, and Cys 80 as the sulfhydryl groups alkylated in these three incompletely oxidized, partially active forms. Thus, it appears that the interatomic interactions maintaining the compact three-dimensional structure of native lysozyme are operational even when one of these three native disulfide bonds between Cys 6 and Cys 127, Cys 76 and Cys 94, and Cys 64 and 80 is open.     

Differences between Saccharomyces cerevisiae and Bacillus subtilis in secretion of human lysozyme

Saccharomyces cerevisiae secreted human lysozyme in the medium as an active form when the signal peptides of chicken lysozyme and a chicken lysozyme-Aspergillus awamori glucoamylase hybrid were used, whereas it did not synthesize any human lysozyme protein by using the signal peptide of A. awamori glucoamylase. The secreted lysozyme was easily purified and crystallized. On the other hand, Bacillus subtilis secreted an inactive human lysozyme, which seemed to have incorrect disulfide bonds, with the signal peptide of amylase and its mutants. The free energy changes for the membrane translocation of the signal peptides are related to the secretion of human lysozyme in S. cerevisiae, but not in B. subtilis. These results indicate that differences exist between S. cerevisiae and B. subtilis in the secretion of human lysozyme.     

Identification of the core structure of lysozyme amyloid fibrils by proteolysis

Human lysozyme variants form amyloid fibrils in individuals suffering from a familial non-neuropathic systemic amyloidosis. In vitro, wild-type human and hen lysozyme, and the amyloidogenic mutants can be induced to form amyloid fibrils when incubated under appropriate conditions. In this study, fibrils of wild-type human lysozyme formed at low pH have been analyzed by a combination of limited proteolysis and Fourier-transform infrared (FTIR) spectroscopy, in order to map conformational features of the 130 residue chain of lysozyme when embedded in the amyloid aggregates. After digestion with pepsin at low pH, the lysozyme fibrils were found to be composed primarily of N and C-terminally truncated protein species encompassing residues 26-123 and 32-108, although a significant minority of molecules was found to be completely resistant to proteolysis under these conditions. FTIR spectra provide evidence that lysozyme fibrils contain extensive beta-sheet structure and a substantial element of non beta-sheet or random structure that is reduced significantly in the fibrils after digestion. The sequence 32-108 includes the beta-sheet and helix C of the native protein, previously found to be prone to unfold locally in human lysozyme and its pathogenic variants. Moreover, this core structure of the lysozyme fibrils encompasses the highly aggregation-prone region of the sequence recently identified in hen lysozyme. The present proteolytic data indicate that the region of the lysozyme molecule that unfolds and aggregates most readily corresponds to the most highly protease-resistant and thus highly structured region of the majority of mature amyloid fibrils. Overall, the data show that amyloid formation does not require the participation of the entire lysozyme chain. The majority of amyloid fibrils formed from lysozyme under the conditions used here contain a core structure involving some 50% of the polypeptide chain that is flanked by proteolytically accessible N and C-terminal regions.     

Molecularly imprinted polymer-assisted refolding of lysozyme

For production of active proteins using heterologous expression systems, refolding of proteins from inclusion bodies often creates a bottleneck due to its poor yield. In this study, we show that molecularly imprinted polymer (MIP) toward native lysozyme promotes the folding of chemically denatured lysozyme. The MIP, which was prepared with 1 M acrylamide, 1 M methacrylic acid, 1 M 2-(dimethylamino)ethyl methacrylate, and 5 mg/mL lysozyme, successfully promoted the refolding of lysozyme, whereas the non-imprinted polymer did not. The refolding yield of 90% was achieved when 15 mg of the MIP was added to 0.3 mg of the unfolded lysozyme. The parallel relationship between the refolding yield and the binding capacity of the MIP suggests that MIP promotes refolding through shifting the folding equilibrium toward the native form by binding the refolded protein.     

Solubilization limit of lysozyme into DODMAC reverse micelles

Dioctyldimethyl ammonium chloride (DODMAC) was used to form reverse micelles and to extract lysozyme from an aqueous solution into an organic phase. The solubilization behavior of lysozyme into a DODMAC reverse micellar phase was examined in terms of the temperature, the type of cations in the aqueous phase, and the surfactant concentration in the organic phase. Complete removal of lysozyme from the aqueous phase was obtained when the pH was set one unit higher than the pI of the protein. However, it was found that there is a solubilization limit of lysozyme in the organic phase. Not all the lysozyme extracted out of the initial aqueous phase was solubilized into the DODMAC reverse micellar phase, resulting in the formation of white precipitate at the aqueous-organic interface. Temperature has a negligible effect on the solubilization limit of lysozyme. The value of the solubilization limit is a strong function of the type of cations present in the aqueous phase, indicating an important role of lysozyme-cation interactions on the extraction process. An increase in the DODMAC concentration from 100-200 mM resulted in little change in the highest concentration of lysozyme obtained in the organic phase.     

Application of zero-length cross-linking to form lysozyme, horseradish peroxidase and lysozyme-peroxidase dimers: Activity and stability

A facile method for the formation of covalent bonds between protein molecules is zero-length cross-linking. This method enables the formation of cross-links without use of any chemical reagents. Here, the cross-linking is performed for lysozyme, peroxidase (a glycoprotein) and between lysozyme–peroxidase by the method of Simons et al. [B.L. Simons, M.C. King, T. Cyr, M.A. Hefford, H. Kaplan, Covalent cross-linking of protein without chemical reagents, Protein Sci. 2002, 11, 1558–1564]. Approximately one-third of the total lysozyme becomes cross-linked and the dimer form was the major product for both enzymes. This modification induced some changes in the kinetic properties of the dimer peroxidase, as evident by two-fold increasing of V_max compared to the monomer but the enzymatic activity of cross-linked lysozyme dimer was the same as monomer. The activity of lysozyme dimer remained constant up to 10 min at 80 °C, while peroxidase activity of both monomer and dimer began to decrease after heating. The structural changes of the enzymes were investigated by circular dichroism and intrinsic fluorescence techniques. Near UV result showed lysozyme possess a compact structure in the dimer form but disruption of tertiary structure of peroxidase dimer was observed. Also conformational changes were detected and discussed by intrinsic fluorescence experiments. Effect of several metals in the formation of lysozyme dimer showed that Co²⁺ is the most effective one but its effect was marginal. At the end formation of heterogeneous dimer, peroxidase–lysozyme, was achieved using this method.     

A structural study of calcium-binding equine lysozyme by two-dimensional 1H-NMR.

Since 1H-NMR spectra of the calcium bound form (holo) and the calcium free form (apo) of equine lysozyme have an overall similarity, the folded structure of apo equine lysozyme seems to be similar to the holo structure at 25 degrees C and pH 7.0, even at low ionic strengths except for subtle conformational change. However, calcium titration experiments showed that a number of resonances change by a slow exchange process. The changes saturated at one calcium ion per one lysozyme molecule, and no more change was observed by further addition of calcium ions. This shows that just one calcium ion binds to equine lysozyme. To make assignments for these changed proton resonances, two-dimensional 1H-NMR studies, correlated spectroscopy (COSY), two-dimensional homonuclear Hartmann-Hahn spectroscopy (HOHAHA) and nuclear Overhauser effect spectroscopy (NOESY) were carried out. A structural model of equine lysozyme based on the crystal structure of human lysozyme was estimated and used to assign some resonances in the aromatic and beta-sheet regions. It was possible to use some proton signals as a probe to determine the specific conformational change induced by calcium ions. The calcium binding constant KCa was estimated from calcium titration experiments in which changes in the proton signal were monitored. The log KCa value was found to be on the order of 6-7, which is in agreement with the calcium binding constant determined by fluorescence probes. This means that the protons are affected by specific calcium binding.     

人溶菌酶基因的合成和克隆

用固相亚磷酰胺法合成了人溶菌酶的全基因,全长为409bp,它包括了编码人溶菌酶的结梅基因,起始密码于ATG,终止密码子TAA、TGA,以及两端的BamHI和SphI的识别顺序。整个基因分成24个寡聚核苷酸片段进行合成,每个片段长度分别为26至38个核苷酸．然后用两种方法酶促连接成完整的人溶菌酶基因。基因克隆到M13载体上。用点杂交和限制酶酶切分析确定阳性克隆株。用双脱氧链终止法进行序列分析,证实所合成的人溶菌酶基因序列与设计的完全一致。     

Specific carbodiimide-binding mechanism for the selective modification of the aspartic acid-101 residue of lysozyme in the carbodiimide-amine reaction

A mechanism for the selective modification of Asp-101 in hen egg-white lysozyme with an amine nucleophile catalyzed by 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride (EDC) was investigated using ethanolamine as a nucleophile at pH 5.0 and room temperature. In the presence of N-acetyl-D-glucosamine (NAG) and its oligomers [(NAG)n, n = 2 and 3] under the conditions with which about 90% of lysozyme was calculated to form complexes, the formation of Asp-101 modified lysozyme decreased markedly but to different degrees, that is (NAG)3 was the most and NAG the least effective. When the lysozyme derivative, in which Trp-62 in the active site cleft was oxidized to oxindolealanine (Ox-62 lysozyme), was used in place of native lysozyme, the formation of Asp-101 modified derivative decreased to about half, which was similar to the decrease in the presence of (NAG)2. In the presence of 0.5 M NaCl, on the other hand, the formation of Asp-101 modified lysozyme was considerably enhanced. From these observations, it is concluded that EDC binds to the active site cleft of lysozyme to specifically activate Asp-101. The affinity of EDC to the active site of lysozyme is partly due to the hydrophobic interaction of EDC with the Trp-62 residue at sub-site B of lysozyme. EDC is an activating reagent for carboxyl groups unlike most active site-directed reagents which produce final products directly. Therefore, the active site-directed nature of EDC was very useful because it made it possible to selectively introduce various amines as needed at a particular carboxyl group of lysozyme.     

Immunocytochemical observations on the organ distribution of exogenous monomeric and dimerized lysozyme.

Using commercial anti-lysozyme antibodies and anti-dimerized lysozyme rabbit serum produced by us we demonstrated by immunohistochemistry, in some organs of rats, the expression of exogenous egg white lysozyme preparations beside the native lysozyme. After oral administration, the egg white lysozyme was detected in intestinal epithelium, proximal and distal tubules of some nephrons, pulmonary alveolar walls and hepatocytes in the 3rd zone of liver acini, whereas native lysozyme was strongly expressed in intestinal and pulmonary macrophages in both the experimental and control animals. However, expression of the dimerized lysozyme released from the intraperitoneally implanted mini-osmotic pumps and detected using specific antisera was evident only on erythrocytes in intestinal blood vessels. It is concluded that the lysozyme preparations administered per os or parenterally are resorbed to blood circulation and distributed among various organs in an active form and maintaining their antigenic specificity. It may speak for their direct anti-inflammatory and immunomodulatory effects in respiratory, urinary, digestive and other systems.     

The temperature and pH-dependent transition of hen lysozyme. Characterization of two temperature-defined domains and of an N-acetylglucosamine (inhibitor)-insensitive form

The previously described temperature and pH-dependent transition in the solid state of hen lysozyme was studied in solution. Experiment concerning the velocity of lysis ofM. luteus by lysozyme and its behavior in presence of an inhibitor (GlcNAc) as well as a reinvestigation of the Arrhenius curves over a large range of pH, demonstrated the existence of two temperature-induced domains. An inhibitor-insensitive lysozyme form was characterized at 40° (physiological temperature).112th communication on lysozymes. Inquiries should be addressed to P. Jollès.     

激光在国外兽医领域应用及基础研究进展

本文主要就激光在国外兽医领域的激光内窥镜外科、肿瘤外科、眼外科、小动物外科以及激光动力学治疗等方面的应用及基础理论方面的研究进展进行综述。     

Two-State Folding of Lysozyme Versus Multiple-State Folding of α-Lactalbumin Illustrated by the Technique of Disulfide Scrambling

The folding of lysozyme and of alpha-lactalbumin exhibits vastly different kinetics and pathways. Existing evidence indicates that folding intermediates of alphaLA form a well-populated equilibrium molten globule state that is absent in the case of hen lysozyme. We demonstrate here such divergent folding mechanisms of lysozyme and alphaLA using the technique of disulfide scrambling. Two extensively unfolded homologous isomers (beads-form) of lysozyme (Cys6-Cys30, Cys64-Cys76, Cys80-Cys94, Cys115-Cys127) and alphaLA (Cys6-Cys28, Cys61-Cys73, Cys77-Cys91, Cys111-Cys120) were allowed to refold in parallel to form the native protein. Folding kinetics was measured by the recovery of the native structure. Folding intermediates, which illustrate the folding pathway, were trapped by quenching disulfide shuffling and were analyzed by reversed-phase high-pressure liquid chromatography. The results revealed that under identical folding conditions, the folding rate of lysozyme is about 30-fold faster than that of alphaLA. Folding intermediates of lysozyme are far less heterogeneous and sparsely populated than those of alphaLA. Numerous predominant on-pathway and off-pathway intermediates observed along the folding pathway of alphaLA are conspicuously absent in the case of lysozyme. The difference is most striking under fast folding conditions performed in the presence of protein disulfide isomerase. Under these conditions, folding of lysozyme undergoes a near two-state mechanism without accumulation of stable folding intermediates.     

Kinetics and equilibria of lysozyme precipitation and crystallization in concentrated ammonium sulfate solutions

The kinetics and thermodynamics of lysozyme precipitation in ammonium sulfate solutions at pH 4 and 8 and room temperature were studied. X-ray powder diffraction (XRD) was used to characterize the structure of lysozyme precipitates. It was found that, if sufficient time was allowed, microcrystals developed following an induction period after initial lysozyme precipitation, even up to ionic strengths of 8 m and at acidic pH, where lysozyme is refractory to crystallization in ammonium sulfate. The full set of precipitation and crystallization data allowed construction of a phase diagram of lysozyme, showing the ammonium sulfate dependence. It suggests that precipitation may reflect a frustrated metastable liquid-liquid phase separation, which would allow this process to be understood within the framework of the generic phase diagram for proteins. The results also demonstrate that XRD, more frequently used for characterizing inorganic and organic polycrystalline materials, is useful both in characterizing the presence of crystals in the dense phase and in verifying the crystal form of proteins.     

Effect of organics on sulfur-utilizing autotrophic denitrification under mixotrophic conditions

Thiol groups were introduced to dermal bovine collagen (DBC) by the reaction with gamma-thiobutyrolactone. Thiolated DBC reacted with 2-pyridyl disulfide group introduced to lysozyme to form DBC-lysozyme conjugate through disulfide bridge. The enzymatic activity of freshly prepared conjugate was almost unchanged during ten consecutive runs over one month. The DBC-lysozyme conjugate showed the maximum activity at pH 6.3, on the contrary, that of native lysozyme was pH 9.0. Thermal stability of lysozyme was enhanced by the conjugation with DBC. The present results showed that the conjugation using thiolated collagen could be one of the useful alternative approaches to modify collagen with bioactive molecules.     

Affinity extraction of proteins with a reversed micellar system composed of Cibacron Blue-modified lecithin

Crude soybean lecithin was used as a novel surfactant to form reversed micelles in n-hexane. Cibacron Blue F-3GA (CB) was directly immobilized to the reversed micelles by a two-phase reaction. The reversed micellar system without CB showed low solubilizing capacity for low molecular weight proteins, lysozyme, and cytochrome c due to the weak electrostatic interactions. The introduction of CB significantly increased the solubilization of lysozyme because of its affinity binding to CB but showed no effect on the solubilization of cytochrome c since it did not bind to CB. Although bovine serum albumin had an affinity for CB, it was not extracted to the reversed micelles containing CB because its high molecular weight resulted in a significant steric hindrance effect. Thus the reversed micellar system had a high selectivity resulting from both biospecific and steric hindrance effects. The extraction yield of lysozyme decreased significantly with increasing ionic strength. Therefore, the back extraction of lysozyme was carried out using a stripping solution with an ionic strength of 0.865 mol/L. The overall recovery yield of lysozyme after back extraction could be increased to 87% by stripping for 2 h. The recovered lysozyme exhibited an activity equivalent to native lysozyme, and its secondary structure was also unchanged.     

Novel function of guanidine hydrochloride in reverse micellar extraction of lysozyme from chicken egg white

The efficiency of guanidine hydrochloride (GuHCl) addition in the suppression of gel formation and the extraction of lysozyme during reverse micellar extraction from chicken egg white was investigated. A low concentration of GuHCl in the feed permitted the successful extraction of lysozyme in its native form without gel formation, which is perceived as a novel function of GuHCl. The highest recovery and specific activity of lysozyme were obtained at a GuHCl concentration of 0.06 M in 25 mM AOT reverse micellar extraction from 20-fold-diluted natural chicken egg white. Lysozyme and ovalbumin CD spectra in the corresponding GuHCl aqueous solutions revealed no changes in the higher order structures of the proteins. Furthermore, the specific activity of lysozyme in the feed was well preserved in the GuHCl system. (c) 1995 John Wiley & Sons, Inc.     

Effect of chemical modifications of tryptophan residues on the folding of reduced hen egg-white lysozyme

The effects of chemical modifications of Trp62 and Trp108 on the folding of hen egg-white lysozyme from the reduced form were investigated by means of the sulfhydryl-disulfide interchange reaction at pH 8 and 40 degrees C. The folding of reduced lysozyme was monitored by following the recovery of the original activity. Under the conditions employed, the apparent first-order rate constant for the folding of reduced lysozyme was not changed by the modifications of both Trp62 and Trp108 and the folding was completed within 30 min. However, the extent of the correct folding was changed by the modification of Trp62 but not by that of Trp108. Native and oxindolealanine108 lysozymes recovered 80 and 81% of their original activities after 30-min refolding, respectively, but Trp62-modified lysozymes recovered their activities to a lesser extent than native and oxindolealanine108 lysozymes. The recovered activities of Trp62-modified lysozymes after 30-min refolding were 63% for oxindolealanine62 lysozyme, 65% for delta 1-carboxamidomethylthiotryptophan62 lysozyme, and 52% for delta 1-carboxymethylthiotryptophan62 lysozyme. These results suggest that Trp62 is important for preventing the misfolding of reduced lysozyme, but that neither Trp62 nor Trp108 is involved in the rate-determining step (the slowest step) in the folding pathway. A decrease in the hydrophobic nature of Trp62 seems to increase the misfolding and thus to decrease the extent of the correct folding of reduced lysozyme. A mechanism for the involvement of Trp62 in the folding pathway of reduced lysozyme is proposed.