Frans_M_Everaerts_Isotachophoresis_378342.pdf

ENZYMATIC CONVERSION OF PYRUVATE 355
TABLE 15.6
COMPOSITION OF THE SOLUTIONS APPLIED FOR THE DETERMINATION OF THE EXTENT
OF CONVERSION
Solution Concentration (mmole/l)
No.
.-
ATP MgSO, Glucose Fructose
1.5
1.5
1.5
1.5
1.5
1.5
2
1.45
0.5
-
TABLE 15.7
OPERATIONAL SYSTEM AT pH 4.7 SUITABLE FOR ANIONIC SEPARATIONS
Solvent: H, 0.
Electric current (PA): Ca. 70-100.
Purification: MES is purified by recrystallization three times from water-ethanol, the
crystals being washed with acetone. e-Aminocaproic acid is purified by
recrystallization.
Electrolyte
Leading Terminating
Anion c1- MES-
Concentration 0.01 N Ca. 0.01 N
Counter ion* HOOC-C, Ha CH, N'H Tris+
and His+
PH 4.5 (c-Aminocaproic Ca. 4.5
acid); 4.7 (His)
Additive 0.05% Polyvinyl None
aIcohol (Mowiol)
*The leading electrolyte is prepared as follows: first, 0.01 N hydrochloric acid; secondly, eaminocaproic
acid is added till a pH value of 4.5 has been reached; and finally, histidine is added till a pH value of
4.7 has been reached.
15.3. ENZYMATIC CONVERSION OF PYRUVATE INTO LACTATE WITH LACTATE
DEHYDROGENASE FROM PIG HEART
This type of enzymatic reaction was chosen because NAD (NADH) is involved. The
reaction is:
LDH
Pyruvate + NADH+ + H+ NAD + lactate* (15.3)
A reaction mixture was prepared by mixing 25 ml of buffer solution (0.01 Nhydrochloric
*At pH 7.5 lactate is formed; at pH 8.9 pyruvate is formed.
-
2
1
0.5