Lund Marianne Nissen, Olsen Karsten, Sørensen John, Skibsted Leif H
Food Chemistry, Department of Food Science, The Royal Veterinary and Agricultural University, Rolighedsvej 30, DK-1958 Frederiksberg C, Denmark.
J Agric Food Chem. 2005 Mar 23;53(6):2095-102. doi: 10.1021/jf048688c.
The lactosylation of alpha-lactalbumin in aqueous solution was followed at pH(c) = 6.0, 6.3, 7.0, 7.3, and 7.9 and constant ionic strength (I = 0.080) at 50-60 degrees C by reversed-phase high-performance liquid chromatography (RP-HPLC) and electrospray mass spectrometry (MS). The rate of the lactosylation reaction increased with increasing pH and with temperature most significantly at lower pH. The rate of lactosylation could be described by an acid dissociation curve corresponding to pK(a) of the epsilon-amino group of lysine in alpha-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 by dividing with the lactose concentration. Second-order rate constants for protonated and unprotonated lysine in alpha-lactalbumin both showed Arrhenius behavior, and using transition-state theory, DeltaH# = 31 +/- 2 kJ/mol and DeltaS# = -266 +/- 48 J/(mol . K) were determined for the unprotonated form and DeltaH# = 158 +/- 49 kJ/mol and DeltaS# = 80 +/- 150 J/(mol . K) for the protonated form, respectively. On the basis of the marked differences in activation parameters, initial formation of a lactosylamine is suggested as rate-determining for reaction between lactose and a protonated lysine in alpha-lactalbumin, while subsequent water elimination to form a Schiff base becomes rate-determining for the unprotonated form.
在50 - 60℃、pH(c) = 6.0、6.3、7.0、7.3和7.9以及恒定离子强度（I = 0.080）的水溶液中，通过反相高效液相色谱（RP - HPLC）和电喷雾质谱（MS）跟踪α - 乳白蛋白的乳糖基化反应。乳糖基化反应速率随pH升高而增加，且在较低pH时随温度升高最为显著。乳糖基化反应速率可用与α - 乳白蛋白中赖氨酸ε - 氨基的pK(a)相对应的酸解离曲线来描述。根据乳糖过量条件下的初始速率计算出伪一级速率常数，并通过除以乳糖浓度进一步转化为二级速率常数。α - 乳白蛋白中质子化和未质子化赖氨酸的二级速率常数均呈现阿累尼乌斯行为，利用过渡态理论，未质子化形式的ΔH# = 31 ± 2 kJ/mol，ΔS# = -266 ± 48 J/(mol·K)，质子化形式的ΔH# = 158 ± 49 kJ/mol，ΔS# = 80 ± 150 J/(mol·K)。基于活化参数的显著差异，推测乳糖胺的初始形成是乳糖与α - 乳白蛋白中质子化赖氨酸反应的速率决定步骤，而随后消除水形成席夫碱则是未质子化形式的速率决定步骤。
