Lactate monooxygenase

W. Trettnak, O.S. Wolfbeis, A fully reversible fiber optic lactate biosensor based on the intrinsic fluorescence of lactate monooxygenase, Fresenius Z. Anal. Chem. 1989, 334, 427. 

LACTATE MONOOXYGENASE LEUCINE KINETICS ACETATE KINASE... 

Two enzymes are commonly used for amperometric biosensors, namely lactate oxidase (LOD) and lactate dehydrogenase (LDH). It should be noted that, in this instance, LOD refers to the enzyme which catalyses the reaction shown in Fig. 23.4, in which the products are pyruvate and H202. This type of enzyme was formerly assigned the E.C. number 1.1.3.2, but this was confused with lactate monooxygenase (E.C. 1.13.12.4), which is also commonly referred to as type I lactate oxidase [55] or simply lactate oxidase [56] whose products are acetate, C02 and H202. The LOD which catalyses the reaction shown in Fig. 23.4 has also been referred to as type II lactate oxidase [55] following clarification of this point in a published letter [57], current publications refer to this enzyme as E.C. 1.1.3.x. 

Owing to differences in the Kyi values and the layer thickness, the transient from kinetic to diffusion control of different enzyme electrodes takes place at rather different enzyme activities. Gelatin-entrapped enzymes exhibit transient values of 0.17 U/cm2 (uricase,iifM= 17 pmol/1), 16 U/cm2 (urease, Kyi = 2 mmol/1) and 1.0 U/cm2 (lactate monooxygenase, Km = 7.2 mmol/1). 

Lactate monooxygenase (LMO, EC 1.13.12.4) from Mycobacterium smegmatis is a flavoprotein with a molecular weight of 340 000 and a -KMflactate) of 8 mmol/1. The enzyme has been immobilized on porous glass and employed in an enzyme thermistor allowing lactate to be measured in the range 0.005-2 mmolA (Danielsson et al., 1981). 

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-... 

Fig. 96. Function of a lactate monooxygenase membrane as anti-interference layer for lactate. The current is a measure of the permeation of lactate through the membrane.

Malate/ oxaloacetate lactate monooxygenase + malate dehydrogenase membrane O2 electrode 3 Schubert et al. (1991)... 

During this coupled reaction in the membrane large amounts of lactate and oxalate are formed which can be indicated by coupled lactate monooxygenase or oxalate oxidase. Using lactate monooxygenase an amplification factor for NADH of 220 was obtained. 

LMO = lactate monooxygenase, LDH = lactate dehydrogenase, PK. = pyruvate kinase, GOD > glucose oxidase, GLOD = glutamate oxidase, (J-gal = (J-galactosidase, GA = glucoamylase... 

Pyruvate kinase (PK) activity in hemolyzed erythrocytes has been determined by using an LDH-lactate monooxygenase sequence electrode (Weigelt et al., 1988). The enzymes were immobilized in gelatin and attached to an oxygen probe. Since the sample material contains only... 

Figure 17.6 (a) The principle of an accumulation biosensor, (b) Current-time curves for a sensor operated without and with intermediate accumulation. The difference in the steady state and peak currents represents the amplification of the sensor response, (c) Glycerol measurement without and with NADH accumulation on an enzyme electrode containing immobilized glycerol dehydrogenase, lactate dehydrogenase, and lactate monooxygenase. NADH is stripped by pyruvate addition after 6 min accumulation (reproduced with the permission of Elsevier Science Publishers BV). 

L-lactate monooxygenase (LMO) catalyzes the reaction of L-lactate with O2, producing acetate, CO2, and H2O (Equation (7)). 

Another promising type of optical affinity sensor has recently been described by IVettnak and Wolfbeis [221, 222]. The fluoresence of flavoproteins glucose oxidase, lactate monooxygenase) changes with the interaction between enzyme and substrate, and this can be used for substrate detection. As the reduced prosthetic group is reoxidized by Oj, the process is reversible, so in this case the affinant is self-regenerating. 

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