Isolation and characterization of human milk lysozyme

Parry, R.M.; Chandan, Ramesh C.; Shahani, K. M.

doi:10.1016/0003-9861(69)90009-5

Cited by 98 publications

(43 citation statements)

References 18 publications

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“…The amino acid composition of human milk lysozyme reported here was not significantly different from those of human milk (4,5,9), leucocyte (5, 10) and skin (1), although they were markedly different from that of egg white lysozyme.…”

Section: Resultscontrasting

confidence: 64%

A Rapid Purification mlthod and Electrophoretic Studies of Human Milk Lysozyme

Kubagawa

Ogawa

1972

Juntendo Medical Journal

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Section: Resultscontrasting

confidence: 64%

A Rapid Purification mlthod and Electrophoretic Studies of Human Milk Lysozyme

Kubagawa

Ogawa

1972

Juntendo Medical Journal

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“…After treatment, atomic absorption spectra confirmed that >90%o of the bound Ca2+ was removed. Concentrations of the lysozymes were determined spectrophotometrically by using el% nm = 25.6 for human lysozyme (13) and its mutant.…”

Section: Methodsmentioning

confidence: 99%

Entropic stabilization of a mutant human lysozyme induced by calcium binding.

Kuroki

Kawakita

Nakamura

et al. 1992

Proc. Natl. Acad. Sci. U.S.A.

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The stabilization mechanism of the mutant human lysozyme with a calcium binding site (D86/92) was investigated by using calorimetric approaches. By differential scanning calorimetry, the enthalpy change (Al) in the unfolding of holo-D86/92 was found to be 6.8 kcal/mol smaller than that of the wild-type and apo-D86/92 lysozymes at 85TC.However, the unfolding Gibbs energy change (AG) of the holo mutant was 3.3 kcal/mol greater than the apo type at 85C, indicating a significant decrease of entropy (TAS = 10.1 kcal/mol) in the presence of Ca2 . Subsequently, the Ca2+ binding process in the folded state of the mutant was analyzed by using titration isothermal calorimetry. The binding enthalpy change was estimated to be 4.5 kcal/mol, and AG was -8.1 kcal/mol at 85°C, which indicates that the binding was caused by a large increase in entropy (TAS = 12.6 kcal/mol). From these analyses, the unfolded holo mutant was determined to bind Ca2+ with a binding AG of -4.8 kcal/mol (Al = -2.6 kcal/mol, TAS = 2.2 kcal/mol) at 85°C. Therefore, the major cause of stabilization of holo-D86/92 is the decrease in entropy of the peptide chain due to Ca2+ binding to the unfolded protein.It is known that ligand binding stabilizes a protein (1-3). This kind of stabilization is considered to be very important in biological systems because the stabilization of a protein can be easily regulated by the concentration of ligand in the system (2, 4). The mechanism of the stabilization is stoichiometrically explained by the shift of the folding-unfolding equilibrium to the folded state caused by the higher affinity of ligand to the folded state (5, 6). However, the stabilization mechanism of a protein induced by ligand binding is not so clear from the thermodynamic point of view. The reactions involve not only the change of protein conformation but also the interaction of ligand with water molecules, which makes it complicated to understand the thermodynamic mechanism of the stabilization induced by ligand binding alone. Therefore, the most effective way to clarify the mechanism is the direct analysis ofboth the denaturation and the ligand binding by using calorimetric approaches in conjunction with information of the tertiary structure of a protein. Two kinds of calorimeters are available for this purpose. One is a differential scanning calorimeter used to obtain thermodynamic parameters of protein unfolding (7,8), and the other is a titration calorimeter used to obtain the thermodynamic parameters of binding of a ligand by a protein (9).A mutant human lysozyme (D86/92) that has an engineered Ca2+ binding site (Ka = 5.0 x 106 M-1) was created by replacing both Gln-86 and Ala-92 with aspartic acid (10). This mutant is a suitable model for the investigation of the stabilization mechanism induced by ligand binding because the x-ray structures of the wild-type (11) and the mutant (12) lysozymes have been already solved at 1.8 A. By using both differential scanning calorimetry (DSC) and isothermal differential titration calorimetry (DTC) it wa...

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“…lysozymes from milk (3,4), tear ( 4 ), saliva (5), placenta ( 6 ), spleen, serum, and leucocytes ( 7 ). Human milk lysozyme has also been isolated by Parry, Chandan and Shahani (8).…”

Section: Introductionmentioning

confidence: 99%