Frans_M_Everaerts_Isotachophoresis_378342.pdf

Chapter 15
Enzymatic reactions
SUMMARY
The possibility of using isotachophoresis for the determination of non-ionic compounds
via enzymatic reactions has been shown experimentally. The method for determining the
initial velocity of an enzymatic reaction is shown, although it is a disadvantage that the
reaction cannot be followed continuously. Two enzymes, representing two different
classes, were chosen arbitrarily: hexokinase and lactate dehydrogenase. In principle, all
enzymatic reactions can be studied in the operational systems specified in ths chapter.
The time of analysis is approximately 15 min from the start of the experiment to
the detection of the last zone.
1 5.1. INTRODUCTION
Isotachophoresis can be applied in many instances to the study of enzymatic reactions,
because ionic constituents are involved. Both enzymatic conversions and kinetics can be
studied. Because enzymatic conversions can be analyzed, many organic substances that
have no or a low effective mobility, e.g., glucose, fructose and urea, can be determined
quantitatively by the isotachophoretic separation technique. While the spectrophoto-
metric detection of enzymatic reactions sometimes needs a second reaction, isotacho-
phoresis can be carried out with a single reaction. Moreover, the purity of the reaction
constituents can be checked before the reaction; the purity of the starting materials is
very important, especially if activities need to be measured [ 11 .
Two types of reactions are considered in this chapter. The choice was made such that
the two types cover all of the main classes of enzymatic reactions. Firstly, the enzymatic
conversion of glucose into glucose-6-phosphate, followed by the conversion of glucosed-
phosphate into gluconate-6-phosphate is discussed. All enzymatic reactions that make use
of ATP, ADP, AMP, NADP and NADPH can be studied*. Secondly, the enzymatic conver-
sion of pyruvate into lactate is discussed, because in this reaction NAD (NADH) is
involved.
A disadvantage if isotachophoresis is applied to the study of enzymatic reactions is
that the reaction cannot be followed continuously, especially if kinetics are being studied.
The analyses were performed in the equipment as described in section 7.4.4.
*Abbreviations used: ADP = adenosine-5’-diphosphate; ATP = adenosine-5’-triphasphate;
LDH = lactate dehydrogenase; MES = morpholinoethanesulphonic acid; NAD = nicotinarnide-adenine
dinucleotide (oxidized); NADH = nicotinamide-adenine dinucleotidc (reduced); NADPH = nicotin-
amide-adenine dinucleo?ide phosphate (reduced); NADP = nicotinamide-adenine dinucleotide
phosphate (oxidized).
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