Buffer-Induced Activation of Calf Intestinal Alkaline Phosphate

Abstract

The influence of Tris, phosphate ions, imidazole, carbonate ions, bicarbonate ions and hydroxyl ions on the rate of activation of calf intestinal alkaline phosphatase after its dissolution in aqueous solution was investigated. The kinetic data are consistent with a general base-catalysed reaction in which the base reacts with an unprotonated enzyme species formed by ionization of a group with pK = 8.9.Mammalian alkaline phosphatases are zinc metallo enzymes catalysing the hydrolysis of phosphomonoesters, and the transfer ofphosphoryl groups to suitable aminoalcohol acceptors. It has long been recognised that dilution of serum, with buffer, has resulted in an activation of alkaline phosphatase [l]. Similarly, reconstitution of lyophilised serum or thawing of frozen serum results in a slow activation of alkaline phosphatase [2,3]. However, this phenomenon has not been satisfactorily characterised. It has been attributed variously to dissociation of partially active lipoprotein complexes [4], reversible association of the enzyme with Tris to form a more active addition complex [5], or from calculations of entropy changes to exposure of additional active sites [6].This paper demonstrates that rapid dissolution of partially purified calf intestinal alkaline phosphatase into buffered solution is followed by an increase in activity, the rate of which depends on pH and buffer concentration. The attendant kinetics of activation are consistent with general base catalysis in which the conjugate base of the buffer activates the enzyme by reaction with an unprotonated species formed by ionisation of a group with pK = 8.9.

Aminoalcohol-Induced Activation of Organophosphorus Hydrolase (OPH) towards Diisopropylfluorophosphate (DFP)

“…Thus, the acceleration of F − releasing in the aminoalcohol buffers might require proper interaction(s) between the hydrogen-bond donors and the hydrophilic leaving groups. The similar activating effects of aminoalcohols have been reported in the BIALP-catalyzed hydrolysis of pNPP with phosphate (Pi) as leaving group [18], also suggesting the specific interaction of aminoalcohols with the hydrophilic leaving groups.…”

“…However, choice of buffer to further enhance OPH activity towards organophosphofluoridates was rarely reported. Previous studies have documented the activation of bovine intestine alkaline phosphatase (EC 3.1.3.1) to degrade p -nitrophenyl phosphate (pNPP) in the presence of aminoalcohol and amine buffers [18–20]. To our knowledge there have been no reports on the effect of reaction buffer on OPH activity towards DFP, a G-type nerve agents analog.…”

“…The alkaline phosphatase (ALP), a typical group of phosphatases (EC 3.1.3.1), exhibited much better performance on phosphomonoesters hydrolysis in the tris and imidazole buffers [18,19]. The various aminoalcohol derivatives, including 2-(ethylamino)ethanol, N -methylethanolamine, diethanolamine, and triethanolamine, have been verified as activators to enhance bovine intestine ALP (BIALP) activity to degrade p -nitrophenyl phosphate (pNPP) [20].…”

Utilization of real‐time electrospray ionization mass spectrometry to gain further insight into the course of nucleotide degradation by intestinal alkaline phosphatase

“…It has been demonstrated in several investigations that diverse factors like type of buffer, ionic strength, metal ions, in particular Mg 2+ and Zn 2+ , may influence activity and specificity of iAP . For instance, it has been shown that the order of degradation of ATP, ADP and AMP is reversed by addition of magnesium .…”

Effects of amines and aminoalcohols on bovine intestine alkaline phosphatase activity

“…Enzyme activity depends on buffer species as well as pH. Bannister and Foster reported that tris, imidazole, phosphate ion, and bicarbonate ion increased BIALP activity [17].…”