Glycation and phosphorylation of α-lactalbumin by dry heating: Effect on protein structure and physiological functions

Electrophoretic patterns of native (N), dry-heated (DH), maltopentaose-conjugated (MP), and phosphorylated, maltopentaose-conjugated (PP−MP) α-LA: (A) native PAGE (15% polyacrylamide gel in the absence of SDS); (B) SDS-PAGE (15% polyacrylamide gel in the presence of 1.7% SDS) with (+) and without (−) 5% of 2-mercaptoethanol (2-ME). Mr=marker protein.

Circular dichroism spectra of native (N), dry-heated (DH), maltopentaose-conjugated (MP), and phosphorylated, maltopentaose-conjugated (PP−MP) α-LA. Protein samples were 0.1g/L in 50mM phosphate buffer (pH 7.0). Circular dichroism spectra of α-LA samples were measured from 190 to 250nm.

Tryptophan fluorescence spectra of native (N), dry-heated (DH), maltopentaose-conjugated (MP), and phosphorylated, maltopentaose-conjugated (PP−MP) α-LA. The excitation wavelength was 280nm, and the emission was scanned from 300 to 400nm. Fluorescence spectra of α-LA samples were measured at 0.1g/L in triplicate.

Differential scanning calorimetry profiles of native (N), dry-heated (DH), maltopentaose-conjugated (MP), and phosphorylated, maltopentaose-conjugated (PP−MP) α-LA. Differential scanning calorimetry scans were performed with a protein solution of 1g/L in 20mM phosphate buffer (pH 7.4). These samples were heated in the calorimeter at a scan rate of 1°C/min over a range of 30 to 80°C.

Antigenicity of native (N), dry-heated (DH), maltopentaose-conjugated (MP), and phosphorylated, maltopentaose-conjugated (PP−MP) α-LA in an adult male Japanese white (JW/CSK) rabbit. The anti-α-LA response after secondary immunization of the rabbit was evaluated by noncompetitive ELISA, and the results are shown as ELISA values (absorbance at 405nm). Each value represents the mean ± SD (n=5). Values with different letters are significantly different at P<0.05 as determined by Student's t-test.

Effect of native (N), dry-heated (DH), maltopentaose-conjugated (MP), and phosphorylated, maltopentaose-conjugated (PP−MP) α-LA on the (A) IL-6 response of THP-1 monocytes and (B) tumor necrosis factor (TNF)-α response of THP-1 macrophages after stimulation with LPS. The control is media with LPS and without protein. Each datum is expressed as a percentage of the control value. Each value shows the mean ± SD (n=5). Values with different letters are significantly different at P<0.05 as determined by Student's t-test.

Calcium phosphate-solubilizing ability of native (N), dry-heated (DH), maltopentaose-conjugated (MP), and phosphorylated, maltopentaose-conjugated (PP−MP) α-LA. The test solution contained 20g/L of protein, 30mM Ca, 22mM inorganic P (Pi), and 10mM citrate, with pH adjusted to 6.7 with 1 M KOH. Each column shows the mean values ± SD (n=3).