Sequence Electrodes for ATP and Glucose-6-phosphate

The combination of the reactions of hexokinase and glucose-6-phos-phate dehydrogenase (G6P-DH)  

Alternatively, glucose-6-phosphate can be determined by using a sequence of alkaline phosphatase (EC 3.1.3.1) and GOD. Such a sequence electrode has been used for the measurement of the competitive inhibitor of phosphatase, inorganic phosphate (see Section 4.4). 

Principle of a sensor containing hexokinase (HK) and glucose-6-phosphate dehydrogenase (G6P-DH) for determination of NADP+, ATP, glucose-6-phosphate, and fructose. 

With a constant of K = 2.7640-5 mol/1 (pH 7.0, 25°C) the equilibrium of the LDH-catalyzed reaction lies far to the lactate side. This means that whereas for lactate sensors based on LDH the forward reaction has to be forced by alkaline buffer and pyruvate- or NADH-trapping agents, the reduction of pyruvate proceeds spontaneously under normal conditions. This direction of the reaction has been used in a sequence electrode for pyruvate assay (Weigelt et al., 1987b). In the presence of lactate monooxygenase (LMO) lactate formed from pyruvate by LDH is oxidized by molecular oxygen, the consumption of which was indicated at a Clark-type electrode. The enzymes were immobilized in a gelatin membrane. Of course such a sensor measures the concentration of lactate in the sample, too. Therefore it is suited to the determination of the lactate/pyruvate ratio, which is a clinically important parameter. Pro- 

The lactate oxidation catalyzed by LMO forms the basis of several other multienzyme electrodes (Fig. 86). The LDH-LMO sensor has also been used to assay the activity of alanine aminotransferase (ALAT, EC 2.6.1.2) and pyruvate kinase (PK, EC 2.7.1.40) (Weigelt 1987 Weigelt et al., 1988). The sample was added to the NADH-containing measuring solution and when the steady state signal for endogenous lactate and pyruvate was attained the substrates of the enzyme to be determined 

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