Glycation and phosphorylation of alpha-lactalbumin by dry heating: effect on protein structure and physiological functions

Alpha-lactalbumin (alpha-LA) was glycated with maltopentaose (MP) through the Maillard reaction (MP-alpha-LA) and subsequently phosphorylated by dry heating in the presence of pyrophosphate to investigate its structure and physiological functions. Glycation occurred effectively, and the sugar content of alpha-LA increased by approximately 22.3% through the Maillard reaction. The phosphorylation of MP-alpha-LA was enhanced with an increase in the dry-heating time from 1 to 5 d, and the phosphorous content of MP-alpha-LA increased by approximately 1.01% by dry heating at pH 4.0 and 85 degrees C for 5 d in the presence of pyrophosphate. The electrophoretic mobility of alpha-LA increased with an increase in the phosphorylation level. The circular dichroism spectra showed that the change in the secondary structure of the alpha-LA molecule by glycation and subsequent phosphorylation was slight. However, the Trp fluorescence intensity was increased by phosphorylation after glycation. In addition, the differential scanning calorimetry thermograms of alpha-LA showed that the denaturation temperature of MP-alpha-LA was decreased by phosphorylation. These results indicated that molten (partially unfolded) conformations of alpha-LA were formed by dry heating in the presence of pyrophosphate after glycation. The anti-alpha-LA antibody response was significantly reduced by glycation and subsequent phosphorylation. The suppressive effect of alpha-LA on the production of proinflammatory cytokines such as IL-6 and tumor necrosis factor-alpha from THP-1 cells after stimulation with lipopolysaccharide was significantly enhanced by glycation with MP and was further enhanced by phosphorylation after glycation. The Ca phosphate-solubilizing ability of alpha-LA was enhanced by phosphorylation. The apoptotic activity of alpha-LA was reduced by glycation and subsequent phosphorylation. These results suggest that phosphorylation by dry heating in the presence of pyrophosphate after glycation with MP through the Maillard reaction is a useful method for improvement of the physiological functions of alpha-LA.