Untitled

A New Approach to the Enzymatic Determination of Magnesium, Calcium, and Barium

“…Previously [11], we found the following optimum conditions for the preparation of the apoform of alkaline phosphatase from seal intestine using EDTA: 0.05 M Tris-HCl buffer solution, pH 9.8; incubation time of the enzyme (E) with the complexone, 15 min; EDTA concentration, 60 µM; and concentration ratio E : EDTA = 1 : 6000.…”

“…On the external addition of zinc and/or magnesium ions to the apophosphatase, the catalytic activity of the enzyme was restored; that is, the reactivation of the enzyme took place. The above reactivation effect formed the basis for the previously developed procedure for determining zinc ions with alkaline phosphatase from seal intestine and EDTA as a chelating agent in the hydrolysis of p-NPP [11]. It seemed reasonable to apply this approach to the determination of magnesium ions because procedures based on its reactivating effect on apophosphatases were not described in the literature, although many researchers observed this effect.…”

“…Procedures for determining Fe(III) with peroxidase [4]; for determining Cu(II) with polyphenol oxidase [5]; and for determining Zn(II) with aminopeptidase [6], alcohol dehydrogenase [7], carboanhydrase [8], and alkaline phosphatase [9][10][11] are well known. A target-oriented change in the sensitivity and selectivity of enzymatic procedures for determining transition metals and heavy metals became possible because of the use of the combined action of two effectors (a metal ion and an organic compound) [12], various analytical signals (reaction rate and the duration of an induction period) [1], various buffer solutions [13], and enzymes from the same class but isolated from different sources [7,12,14].Enzymes have been used very rarely for determining magnesium and alkaline earth metals, although magnesium is a constituent of alkaline phosphatases and ATPases (it is responsible for the high stability of enzymes in storage and regulates (modulates) the catalytic activity of biocatalysts), whereas calcium is a constituent of peroxidases, α -amylases, and phytases (it stabilizes their structures).An analysis of data published in the last decade demonstrated that only a few procedures for the enzymatic determination of the above metals are known. Thus, for example, a highly sensitive procedure was developed for determining magnesium (2.4-2400 µ g/mL); it is based on the activation of firefly luciferase immobilized on BrCN-activated Sepharose [15].…”

“…These procedures are based on the reactivation of apoenzymes. Procedures for determining Fe(III) with peroxidase [4]; for determining Cu(II) with polyphenol oxidase [5]; and for determining Zn(II) with aminopeptidase [6], alcohol dehydrogenase [7], carboanhydrase [8], and alkaline phosphatase [9][10][11] are well known. A target-oriented change in the sensitivity and selectivity of enzymatic procedures for determining transition metals and heavy metals became possible because of the use of the combined action of two effectors (a metal ion and an organic compound) [12], various analytical signals (reaction rate and the duration of an induction period) [1], various buffer solutions [13], and enzymes from the same class but isolated from different sources [7,12,14].…”

Potentialities and prospects for the use of alkaline phosphatases for determining metal ions

“…This method is very efficient, but rather expensive because of the large consumption of enzyme. This explains why dialysis is most widely used [50] as before, although the procedure is time-consuming and multistage.…”