title: Kinetics and mechanism of lactosylation of alpha-lactalbumin.
abstract: 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.
authors.affiliation: Food Chemistry, Department of Food Science, The Royal Veterinary and Agricultural University, Rolighedsvej 30, DK-1958 Frederiksberg C, Denmark.
authors.forename: Marianne Nissen
authors.initials: MN
authors.lastname: Lund
authors.forename: Karsten
authors.initials: K
authors.lastname: Olsen
authors.forename: John
authors.initials: J
authors.lastname: Sørensen
authors.forename: Leif H
authors.initials: LH
authors.lastname: Skibsted
mesh_terms: D002851:Chromatography, High Pressure Liquid; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007768:Lactalbumin / Q000737:chemistry*; D007785:Lactose / Q000737:chemistry*; D008894:Milk Proteins / Q000737:chemistry; D012996:Solutions; D021241:Spectrometry, Mass, Electrospray Ionization; D013816:Thermodynamics; D014867:Water; D000067816:Whey Proteins
chemical_list: D008894:Milk Proteins; D012996:Solutions; D000067816:Whey Proteins; D014867:Water; D007768:Lactalbumin; D007785:Lactose