Milk Lysozyme Research Articles

Equilibrium and kinetic studies on folding of canine milk lysozyme.

Thermal and chemical unfolding studies of the calcium-binding canine lysozyme (CL) by fluorescence and circular dichroism spectroscopy show that, upon unfolding in the absence of calcium ions, a very stable equilibrium intermediate state is formed. At room temperature and pH 7.5, for example, a stable molten globule state is attained in 3 M GdnHCl. The existence of such a pure and stable intermediate state allowed us to extend classical stopped-flow fluorescence measurements that describe the transition from the native to the unfolded form, with kinetic experiments that monitor separately the transition from the unfolded to the intermediate state and from the intermediate to the native state, respectively. The overall refolding kinetics of apo-canine lysozyme are characterized by a significant drop in the fluorescence intensity during the dead time, followed by a monoexponential increase of the fluorescence with k = 3.6 s(-1). Furthermore, the results show that, unlike its drastic effect on the stability, Ca(2+)-binding only marginally affects the refolding kinetics. During the refolding process of apo-CL non-native interactions, comparable to those observed in hen egg white lysozyme, are revealed by a substantial quenching of tryptophan fluorescence. The dissection of the refolding process in two distinct steps shows that these non-native interactions only occur in the final stage of the refolding process in which the two domains match to form the native conformation.

Effect of exercise on immunologic factors in breast milk.

Although it is well documented that breast milk provides optimal nutrition and immune benefits to the infant, factors that influence the immunologic composition of breast milk are less understood. A recent study reported that immunoglobulin A (IgA) levels in breast milk are lower after exercise compared with resting concentrations. However, the women exercised until exhaustion. The effect of moderate exercise on immunologic components in breast milk has not been reported. Therefore, the purpose of this study was to 1) compare the levels of immunologic compounds in breast milk of exercising women with the milk of sedentary women and 2) determine whether 30 minutes of moderate exercise affects immunologic properties of breast milk. Exclusively lactating women were studied at 3 months' postpartum. Women in the exercise group (EG; n = 29) reported exercising aerobically at least 30 minutes/d for 3 days/wk, and women in the sedentary group (SG; n = 24) had exercised once a week or less during the previous 6 weeks. Cardiovascular fitness levels and concentrations of IgA, lactoferrin, and lysozyme in milk were measured. A subsample of the EG (n = 17) participated in a 30-minute exercise session at 75% of maximum heart rate and a rest session of 30 minutes of sitting rest on 2 separate days. Breast milk samples were collected before and 10 and 60 minutes after exercise and rest sessions. IgA, lactoferrin, and lysozyme concentrations were measured. Women in the EG had a higher level of cardiovascular fitness than women in the SG (39.7 +/- 1.0 vs 32.4 +/- 1.0 mL O(2)/kg/min). Milk concentrations of IgA, lactoferrin, or lysozyme were not significantly different between groups. In addition, there were no significant differences in the concentrations of IgA, lactoferrin, or lysozyme after moderate exercise compared with sitting rest. Moderate exercise during lactation improves cardiovascular fitness without affecting levels of IgA, lactoferrin, or lysozyme in breast milk.

Folding mechanism of canine milk lysozyme studied by circular dichroism and fluorescence spectroscopy

We have studied the guanidine hydrochloride‒induced equilibrium unfolding and the kinetics of refolding of canine milk lysozyme by circular dichroism and fluorescence spectroscopy. The thermodynamic analysis of the equilibrium unfolding measured by circular dichroism and fluorescence has shown that unfolding is represented by a three‒state mechanism and that the intermediate state of canine milk lysozyme is remarkably more stable than the intermediates observed in other lysozyme and α-lactalbumin. In the kinetic refolding of this protein, there are at least two kinetic intermediates; a burst=phase intermediate accumulated within the dead time (4 ms) of the measurement and an intermediate that has been observed during the kinetics with a rate constant of 10–20 s–1after the burst phase. This result is apparently in contrast with those previously observed in the kinetic refolding of α‒lactalbumin and equine lysozyme that show only the burst‒phase intermediate. The relationship between the extraordinarily stable equilibrium molten globule and the kinetic folding intermediates will be discussed.

Purification, characterization, antibacterial activity and N-terminal sequencing of buffalo-milk lysozyme.

Lysozyme from buffalo milk was purified to homogeneity and its N-terminal amino acid sequence, biochemical properties and antibacterial spectrum were determined. The purification procedure, comprising ion-exchange chromatography using CM-cellulose and size-exclusion chromatography using Sephadex G-50, conferred 8622-fold purification and 39.3% recovery of lysozyme. The purified enzyme migrated as a single band on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and native PAGE. Immunological purity of lysozyme preparation was confirmed by immuno-electrophoresis. Molecular weight of buffalo-milk lysozyme as determined by SDS-PAGE was 16 kDa and its amino acid composition was determined by reverse phase high performance liquid chromatography (HPLC). The sequence of 23 amino acid residues at the N-terminal end showed 56.5% homology with bovine milk lysozyme and 30.4% with equine milk lysozyme. The specific activity of buffalo milk lysozyme was ten-times that of bovine milk lysozyme. Buffalo-milk lysozyme was active over a wide range of pH and its activity was strongly influenced by molarity of the medium. Antibacterial activity of buffalo-milk lysozyme was determined against 11 species of bacteria; out of seven Gram-positive bacteria tested, four were inhibited, while Gram-negative bacteria were resistant.

Lysozyme Activity in Buffalo Milk: Effect of Lactation Period, Parity, Mastitis, Season in India, pH and Milk Processing Heat Treatment

Lysozyme activity in buffalo milk in relation to the period of lactation, parity of animal, weather conditions and udder infections was studied. Effect of storage and heat processing of milk on lysozyme activity was determined. Lysozyme activity was higher in buffalo milk than in cow milk. Buffalo colostrum showed lysozyme activity 5 times of that in mature milk. Lysozyme activity in buffalo milk was not influenced by the parity of animal and the stage of lactation, however, it increased during extreme whether conditions (winter and summer). Lysozyme in both cow and buffalo milk exhibited maximum activity at pH 7.4. Buffalo milk lysozyme was fully stable while the cow milk lysozyme was partly inactivated by pasteurization (low temperature-long time as well as high temperature-short time treatments). Lysozyme in buffalo milk was more stable than in cow milk during storage and heat treatment. A 10 to 50-fold increase in milk lysozyme activity was observed in mastitic cows. An assay of lysozyme activity in milk can be used to diagnose mastitis in cattle but not in buffaloes. Some buffaloes exhibited 1000 fold greater lysozyme activity and moderately raised somatic cell count in milk, but there was no sign of mastitis in these animals. A possible role of milk lysozyme in prevention of mastitis in buffaloes is discussed.

3M1045 CDおよび蛍光を用いたイヌミルクリゾチームのフォールディング機構の解析(1.蛋白質(C)物性,一般講演,日本生物物理学会第40回年会)

3M1045 CDおよび蛍光を用いたイヌミルクリゾチームのフォールディング機構の解析(1.蛋白質(C)物性,一般講演,日本生物物理学会第40回年会)

Energetics of three-state unfolding of a protein: canine milk lysozyme.

Thermodynamics of thermal transitions of a calcium-binding lysozyme, canine milk lysozyme (CML), was studied using differential scanning calorimetry and compared with those for homologous proteins, human alpha-lactalbumin (alpha-hLA) and equine milk lysozyme (EML). The results showed that CML and EML exhibit two clear heat absorption peaks in the absence of calcium ions (apo-form), although the cooperative thermal transition of alpha-hLA is apparently absent in this form. The first peak represents the unfolding transition from the native to an unfolding intermediate state (N-I transition) and the second peak represents that from the intermediate to the thermally unfolded state (I-U transition). We interpret that the cooperative thermal transition, which is observed between the intermediate and the thermally unfolded states of CML and EML, comes from the native-like packing interaction in their intermediate states. Furthermore, to examine the role of the stabilization mechanism of CML intermediate, we constructed four variant CMLs (H21G, I56L, A93S and V109K), in which the residues of CML are substituted for those of EML, and also investigated their thermal stability. Especially the His21 and Val109 of CML play a role in stabilization of the intermediate state and their contributions to the unfolding free energy are estimated to be 2.0 and 1.8 kJ/mol, respectively. From the results of the mutational analysis, a few differences in the local helical interactions within the alpha-domain are found to be predominant in stabilizing the intermediate state.

3P023イヌミルクリゾチームおよびヒトα-ラクトアルブミンのフォールディングの中間状態における体積挙動

3P023イヌミルクリゾチームおよびヒトα-ラクトアルブミンのフォールディングの中間状態における体積挙動

Dynamics of the Main Immunologically and Nutritionally Available Proteins of Human Milk during Lactation

Microparticle-enhanced nephelometric immunoassays were specifically developed for the quantification ofα -lactalbumin, β -casein, serum albumin, lactoferrin, and lysozyme in human milk. These components, immunoglobulin A and total proteins were assayed in 780 samples collected from 79 mothers during the first 12 weeks of lactation. The data obtained provide a precise description of the dynamics of human milk evolution over time and allow to depict six periods. In the early post-partum period (days 1–4), colostrum contains principally immunological components. The relative concentrations of immunologically and nutritionally available proteins are reversed during the following short transition period (days 5–8). Milk then secreted (days 9–18 and 19–28) appears to have principally a nutritious function. During the two following periods (days 28–49 and 50–84), milk appears well balanced in its nutritional and immunological components, supplying breast-fed infants with appropriate food intake for growth and development and passively protecting them by reincreasing concentrations of immunological components.

Structure and thermodynamics of the extraordinarily stable molten globule state of canine milk lysozyme.

Here, we show that an unfolded intermediate of canine milk lysozyme is extraordinarily stable compared with that of the other members of the lysozyme-alpha-lactalbumin superfamily, which has been studied previously. The stability of the intermediate of this protein was investigated using calorimetry, CD spectroscopy, and NMR spectroscopy, and the results were interpreted in terms of the structure revealed by X-ray crystallography at a resolution of 1.85 A to an R-factor of 17.8%. On the basis of the results of the thermal unfolding, this protein unfolds in two clear cooperative stages, and the melting temperature from the intermediate to the unfolded states is about 20 degrees C higher than that of equine milk lysozyme. Furthermore, the (1)H NMR spectra of canine milk lysozyme at 60 degrees C, essentially 100% of which exists in the intermediate, showed that small resonance peaks that arise from ring-current shifts of aliphatic protons are still present in the upfield region from 0 to -1 ppm. The protein at this temperature (60 degrees C) and pH 4.5 has been found to bind 1-anilino-naphthalene-8-sulfonate (ANS) with enhancement of the fluorescence intensity compared with that of native and thermally unfolded states. We interpret that the extraordinarily stable intermediate is a molten globule state, and the extraordinary stabilization of the molten globule state comes from stronger protection around the C- and D-helix of the aromatic cluster region due to the His-21 residue. The conclusion helps to explain how the molten globule state acquires its structure and stability.

A sensitive HPLC method to detect hen's egg white lysozyme in milk and dairy products

Because of the lytic activity on the cell wall of bacteria like Clostridium tyrobutyricum, hen's egg white lysozyme is used in cheese manufacturing to prevent late blowing. A HPLC method capable to quantify as low as 0.8 ppm lysozyme in milk and cheese is proposed. Lysozyme was extracted with 1 m NaCl at pH 6.0 and the extract was deproteinized at low pH values, reaching a recovery up to 90%. Reversed-phase HPLC was performed on a polymeric column and monitoring lysozyme under fluorescence detection (excitation at 280 nm and emission at 340 nm). The repeatability of this determination tested on a 15-month-aged hard cheese and expressed as relative standard deviation was 1.47 (n=6) and no interference of peptides formed during ripening was observed. Four commercial preparations of egg white lysozyme gave a similar fluorescence response and the partitioning over cheese and whey was studied with one of them. About 80% of the lysozyme added to the cheesemilk at concentrations up to 80 ppm was retained in the cheese and the concentration factor of lysozyme from the cheesemilk to the cheese proved to be 8.2 on average. This HPLC method and the microbiological assay using Micrococcus luteus were compared in cheese analysis, proving the former to be more accurate and reliable than the latter. Eighteen commercial samples including both generic cheeses and cheeses having protected designation of origin, all of them not declared to contain lysozyme, showed concentrations of this enzyme ranging from 0 to 111 ppm.

Hydrogen exchange study of canine milk lysozyme: stabilization mechanism of the molten globule.

The native state (1)H, (15)N resonance assignment of 123 of the 128 nonproline residues of canine milk lysozyme has enabled measurements of the amide hydrogen exchange of over 70 amide hydrogens in the molten globule state. To elucidate the mechanism of protein folding, the molten globule state has been studied as a model of the folding intermediate state. Lysozyme and alpha-lactalbumin are homologous to each other, but their equilibrium unfolding mechanisms differ. Generally, the folding mechanism of lysozyme obeys a two-state model, whereas that of alpha-lactalbumin follows a three-state model. Exceptions to this rule are equine and canine milk lysozymes, which exhibit a partially unfolded state during the equilibrium unfolding; this state resembles the molten globule state of alpha-lactalbumin but with extreme stability. Study of the molten globules of alpha-lactalbumin and equine milk lysozyme showed that the stabilities of their alpha-helices are similar, despite the differences in the thermodynamic stability of their molten globule states. On the other hand, our hydrogen exchange study of the molten globule of canine milk lysozyme showed that the alpha-helices are more stabilized than in alpha-lactalbumin or equine milk lysozyme and that this enhanced stability is caused by the strengthened cooperative interaction between secondary structure elements. Thus, our results underscore the importance of the cooperative interaction in the stability of the molten globule state.

3A0915 イヌミルクリゾチームの折り畳み中間体およびモルテングロビュール状態の重水素交換反応による解析

3A0915 イヌミルクリゾチームの折り畳み中間体およびモルテングロビュール状態の重水素交換反応による解析

1A1645 イヌ・ミルク・リゾチームの精製とフォールディング機構の研究

1A1645 イヌ・ミルク・リゾチームの精製とフォールディング機構の研究

Immunological and nutritional composition of human milk in relation to prematurity and mother's parity during the first 2 weeks of lactation.

To investigate the effect of prematurity and parity on the dynamics of the major immunologic and nutritional proteins of human milk over the first 2 weeks of lactation. Microparticle-enhanced nephelometric immunoassays were developed for the quantification of alpha-lactalbumin, beta-casein, serum albumin, lactoferrin, and lysozyme in human milk. These components, immunoglobulin A, and total proteins were assayed in 368 individual samples collected from 74 mothers. The dynamics of the major immunologic and nutritional proteins in early lactation presented similar patterns in preterm and term human milks. In comparison with term milk, preterm milk was globally characterized by higher concentrations of immune proteins and lower concentrations of nutritive proteins. These differences were increased by the degree of prematurity, which, however, influenced the absolute and relative protein concentrations differently, depending on the stage of lactation. The protein composition of term milk was similar, whatever the mother's parity. Conversely, the influence of prematurity on the levels of milk proteins during the first days of lactation was even greater in primiparous mothers. This precise description of the composition of preterm and term milk, regarding the main nutritional and immunologic proteins, confirms the influence of both prematurity and parity on milk components and demonstrates the combined effect of these two conditions.

Expression of a synthetic gene encoding canine milk lysozyme in Escherichia coli and characterization of the expressed protein.

A high-expression plasmid of the canine milk lysozyme, which belongs to the family of calcium-binding lysozymes, was constructed in order to study its physico-chemical properties. Because the cDNA sequence of the protein has not yet been determined, a 400 base-pair gene encoding canine milk lysozyme was first designed on the basis of the known amino acid sequence. The gene was constructed by an enzymatic assembly of 21 chemically synthesized oligonucleotides and inserted into an Escherichia coli expression vector by stepwise ligation. The expression plasmid thus constructed was transformed into BL21(DE3)/pLysS cells. The gene product accumulated as inclusion bodies in an insoluble fraction. Recombinant canine milk lysozyme was obtained by purification and refolding of the product and showed the same characteristics in terms of bacteriolytic activity and far- and near-UV circular dichroism spectra as the authentic protein. The NMR spectra of refolded lysozyme were also characteristic of a native globular protein. It was concluded that recombinant canine milk lysozyme was folded into the correct native structure. Moreover, the thermal unfolding profiles of the refolded recombinant lysozyme showed a stable equilibrium intermediate, indicating that the molten globule state of this protein was extraordinarily stable. This expression system of canine milk lysozyme will enable biophysical and structural studies of this protein to be extended.

2PA076 イヌミルクリゾチームのモルテングロブュール状態に関する蛋白質工学的研究

2PA076 イヌミルクリゾチームのモルテングロブュール状態に関する蛋白質工学的研究

Calcium-binding and structural stability of echidna and canine milk lysozymes.

For echidna and canine milk lysozymes, which were presumed to be the calcium-binding lysozymes by their amino acid sequences, we have quantitated their calcium-binding strength and examined their guanidine unfolding profiles. The calcium-binding constants of echidna and canine lysozymes were determined to be 8.6 x 10(6) M(-1) and 8.9 x 10(6) M(-1) in 0.1 M KCl at pH 7.1 and 20 C, respectively. The unfolding of decalcified canine lysozyme proceeds in the same manner as that of alpha-lactalbumin, through a stable molten globule intermediate. However, neither calcium-bound nor decalcified echidna lysozyme shows a stable molten globule intermediate. This unfolding profile of echidna lysozyme is identical to that of conventional lysozymes and pigeon egg-white lysozyme, avian calcium-binding lysozyme. This result supports the suggestion of Prager and Jolles (Prager EM, Jolles P. 1996. Animal lysozymes c and g: An overview. In: Jolles P, ed. Lysozymes: Model enzymes in biochemistry and biology. Basel-Boston-Berlin: Birkhauzer Verlag. pp 9-31) that the lineage of avian and echidna calcium-binding lysozymes and that of eutherian calcium-binding lysozymes diverged separately from that of conventional lysozymes.

Microparticle-enhanced nephelometric immunoassay of lysozyme in milk and other human body fluids

Quantitation of lysozyme in human milk was performed by a microparticle-enhanced nephelometric immunoassay based on the measurement of the light scattered during the competitive immunoagglutination of a microparticle-lysozyme conjugate with an anti-lysozyme antiserum. This immunoassay has a detection limit of 8 microg/L of reaction mixture and can be performed using diluted milk (1:6000, in reaction mixture), excluding sample pretreatment. Human milk lysozyme can be quantified over the concentration range 0.09-1.50 g/L, with within- and between-run coefficients of variation <5%. Changes in the lysozyme concentration of human milk during lactation were determined in 636 samples. Lysozyme concentrations (mean +/- SE) decreased from colostrum (0.36 +/- 0.02 g/L) to transitional milk (0.30 +/- 0.01 g/L) and mature milk during days 15-42 (0.30 +/- 0.01 g/L), then increased in the mature milk during days 43-56 (0.35 +/- 0.01 g/L) and especially during days 57-84 (0.83 +/- 0.05 g/L). The proportion of lysozyme contributing to total protein was found to rise during lactation and was as follows: colostrum (1.7%), transitional milk (2.3%), and mature milk from days 15-28 (2.7%), days 29-42 (3.1%), days 43-56 (3.8%), and days 57-84 (7.3%). The assay developed for milk was also suitable for the determination of lysozyme in other human body fluids.

Structure of the calcium-binding echidna milk lysozyme at 1.9 Å Resolution. Erratum

Owing to a printer's error, pages 359 and 360 of this article [Guss, Messer, Costello, Hardy & Kumar (1997). Acta Cryst. D53, 355–363Acta Cryst. D53, 355–363] were incorrectly numbered and thus printed in the wrong order. Revised offprints for this paper are available through the Managing Editor, International Union of Crystallography, 5 Abbey Square, Chester CH 1 2HU, England.