Effects of Oxygen Ingress on Enzyme Kinetic Detection

Concerning kinetic parameters of enzyme kinetics or respirometry measurements, oxygen ingress into the sample can lead to incorrect results. In an oxygen-consuming enzyme reaction like the oxidation of glucose by GOx, oxygen entering the sample partially compensates the oxygen consumption and leads to lower apparent enzyme activities. The same is true for oxygen consumption by respiratory activity of bacteria.

The consequences of oxygen ingress into the sample using oxygen sensors is shown considering the oxidation of glucose by various activities of GOx as example.

Using 100 µL paraffin oil as plate sealing, oxygen ingress into the sample is rather high because it is not only driven by diffusion but forced by convection. This high oxygen ingress partly compensates the oxygen consumption of the enzyme reaction depending on its oxygen uptake rate (OUR). Whereas the kinetics using high GOx activities converge to zero (Fig. 3.13 E, F), lower enzyme activities are more

Chapter 3: Oxygen Ingress into Microtiterplates and its Effect on Kinetic Parameters

influenced by oxygen ingress: After initial oxygen decrease, a steady-state between oxygen ingress into the sample and oxygen consumption by the enzyme kinetic is formed, its level depending on both the enzyme activity and the amount of oxygen ingress. After 120 min, the measurement interval was changed from 30 s to 2 min, which leads to a lower oxygen ingress and lowers the level of the steady state. After some time, however, pO2 starts to increase, which indicates deactivation of the GOx either directly due to destruction

by oxygen [15], or indirectly by H2O2 due to deactivation of catalase [14]. The point in time of this increase depends on the applied total enzyme activity:

Whereas the kinetic using the lowest activity (Fig. 3.13 A) starts to increase even within 2 h, higher activities lead to a later increase (B, C), or the enzyme destruction is even negligible compared to the total enzyme activity (D - F).

The enormous influence of

the oxygen ingress using permeable sealings was further illustrated by a comparative experiment using the determination of the product H2O2 by ABTS instead of the detection of the educt oxygen. The same enzyme activities, glucose concentration and plate sealing (100 µL paraffin oil) were used for both experiments. The increase of absorbance of ABTS by production of H2O2 by GOx was converted into µmol H2O2

using a calibration curve (see Fig. 3.7, right). The formation of H2O2 equals the consumption of the same amount of oxygen (see page 89). More than the 6-fold amount of oxygen diffuses into the sample within 2 h and is consumed by the enzyme reaction (Fig. 3.14, left). After that time, the indicator dye ABTS is completely consumed and the kinetic converges towards an end value. The kinetics involving the two highest enzyme activities (0.1 U and 0.25 U) show a slightly different behaviour:

0

change of interval time from 30 s to 2 min

glucose exhaustion B

C E F D

Fig. 3.13. Oxygen decrease due to the oxidation of 30 mM glucose by different amounts of GOx: (A) 0.004, (B) 0.01, (C) 0.02, (D) 0.04, (E) 0.05, (F) 0.1 U/well. The MTP was sealed with 100 µL of paraffin oil.

Chapter 3: Oxygen Ingress into Microtiterplates and its Effect on Kinetic Parameters

the reaction, or that the H2O2 production is faster than its reaction with ABTS, which decreases during the reaction, leading to a destruction of GOx by excess H2O2. Substrate limitation was ruled out as possible reason due to comparison with the oxygen determination with the oxygen sensor (Fig. 3.14, right). Glucose exhaustion would lead to rapid oxygen increase because the oxygen ingress dominates over the consumption (see Fig. 3.13, F). The determination of the enzyme activity via a parameter independent of oxygen shows the great influence of oxygen ingress on the results obtained with oxygen sensors. Thus, using the initial slope as parameter for the kinetic determination of enzyme activities, oxygen sensors in permeable PS MTPs are not suitable. However, the formation of a steady-state between oxygen consumption and ingress can be used for an end-point determination of the GOx activity. At known and reproducible oxygen ingress, even fast activities, which would be difficult to detect kinetically, can be obtained.

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Fig. 3.14. Oxygen consumption due to the oxidation of 30 mM glucose by different amounts of GOx: 0.002, 0.005, 0.01, 0.02, 0.025, 0.05 U/well. The MTPs were sealed with 100 µL of paraffin oil. Left: Calculated oxygen consumption detected by absorbance measurement using ABTS. Right: Detection using the oxygen-sensitive MTP OxoPlate.

The influence of the plate sealing was investigated by glucose oxidation using 0.5 U GOx/mL in differently covered samples in a PS MTP. The value after 240 min was compared to the same enzyme kinetic measured in a hermetically sealed glass MTP covered with PET foil. Fig. 3.15, right, shows the deviations in c(O2) measured with the PS MTP at the time point (240 min) when the oxygen content is almost zero in the glass MTP well. Without any cover, the deviation of 82 µM from the correct value

Chapter 3: Oxygen Ingress into Microtiterplates and its Effect on Kinetic Parameters

is almost one third of air saturation, with 50 µL of paraffin oil cover it is still 21 µM, whereas all 3 tested foil covers and paraffin wax with and without solvent minimise the deviation. The remaining deviation of 5 µM is therefore due to the oxygen diffusion through the PS MTP material, which has direct contact to the oxygen sensor located at the bottom of the plate.

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Fig. 3.15. Oxygen concentration at the enzymatic oxidation of glucose using 0.5 U/mL GOx in differently sealed PS plates. Left: Kinetics over 4 h; Right: Oxygen content after 240 min. At this time, c(O2) in the hermetically sealed reference glass plate is less than 0.6 µM.