| Abstract
| - The lactosylation of α-lactalbumin in aqueous solution was followed at pHc = 6.0, 6.3, 7.0, 7.3, and7.9 and constant ionic strength (I = 0.080) at 50−60 °C by reversed-phase high-performance liquidchromatography (RP-HPLC) and electrospray mass spectrometry (MS). The rate of the lactosylationreaction increased with increasing pH and with temperature most significantly at lower pH. The rateof lactosylation could be described by an acid dissociation curve corresponding to pKa of the ε-aminogroup of lysine in α-lactalbumin. From initial rates for conditions of excess of lactose, pseudo-first-order rate constants were calculated and further transferred into second-order rate constants bydividing with the lactose concentration. Second-order rate constants for protonated and unprotonatedlysine in α-lactalbumin both showed Arrhenius behavior, and using transition-state theory, ΔH# = 31± 2 kJ/mol and ΔS# = −266 ± 48 J/(mol · K) were determined for the unprotonated form and ΔH#= 158 ± 49 kJ/mol and ΔS# = 80 ± 150 J/(mol · K) for the protonated form, respectively. On thebasis of the marked differences in activation parameters, initial formation of a lactosylamine issuggested as rate-determining for reaction between lactose and a protonated lysine in α-lactalbumin,while subsequent water elimination to form a Schiff base becomes rate-determining for theunprotonated form. Keywords: α-Lactalbumin; lactosylation; pH-effect; activation parameters
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