Frans_M_Everaerts_Isotachophoresis_378342.pdf

348 ENZYMATIC REACTIONS
1 5.2. ENZYMATIC CONVERSION OF GLUCOSE (FRUCTOSE) INTO GLUCOSE-6-
PHOSPHATE (FRUCTOSE-6-PHOSPHATE) WITH HEXOKINASE FROM YEAST
Many papers have dealt with the enzymatic conversion of glucose and fructose into
glucose-6-phosphate and fructose-6-phosphate by hexokinase (e.g., refs. 2-4). Apart
from ATP and the enzyme hexokinase, the reaction can be performed only if sufficient
Mgz+ or Ca2+ is present. Singly charged ions mostly inhibit the reaction [5] . Mg2+ forms
a suitable complex with ATP, but it is beyond the scope of this book to go into too much
detail concerning the complexity of the enzyme reaction itself.
Kinetics and conversions are commonly studied via analyses of the substrate and
product concentrations, especially the changes that occur during the reaction in the
former instance. This is often effected by utilizing a physical property of one of the
reaction constituents: the UV absorption at an appropriate wavelength. This measure-
ment gives all necessary qualitative and quantitative information and is very sensitive. The
reaction discussed briefly in this section, however, can be studied only if it is followed
by a second reaction because ATP and ADP have almost identical UV spectra and
glucose-6-phosphate and glucose have negligible UV absorption. The overall reaction can
be expressed as follows:
hexokinase
Glucose + ATP glucosed-phosphate + ADP
G6PDH
Glucose-6-phosphate + NAPD 6-phosphogluconate + NADPH
(15.1)
(15.2)
The difference in UV absorption between the ions NADP and NADPH at 340 nm gives
information about the conversion of glucose given in eqn. 15.1.
In principle, by means of isotachophoresis all ionic constituents can be determined,
both qualitatively and quantitatively. Suitable operational systems are listed in Tables 15.1
and 15.2. The conditions listed in Table 15.1 are used for measurements at ‘hgh’ concen-
TABLE 15.1
OPERATIONAL SYSTEM AT pH 3.8 SUITABLE FOR ANIONIC SEPARATIONS
Solvent: H, 0.
Electric current @A): Ca. 50-70.
Purification: The buffer was purified by recrystallization in water-ethanol, the crystals
being washed with acetone. The terminator was purified by recrystallization.
Electrolyte
Leading Terminating
Anion c1- p NH, -C, H, -COO-
Concentration 0.01 N Ca. 0.01 N
Counter ion pAla+ H+
PH 3.8 r4
Additive 0.05% Polyvinyl None
alcohol (Mowiol)