8-5
7-5
8-0
7-0
The ra te of th e re a c tio n ( A E340 ^ min”1) b efo re th e a d d itio n of the s u b s tra te "( the b la n k re a c tio n ) was measured in b u ffe r s o lu tio n s o f v ary in g pH v a lu e s .
PROTOCOL
Reagent C o n cen tratio n Volume (cm^] G lycylglycine b u ffe r 0.1 mol 1“ 1 2.05
2-o x o g lu ta ra te 0.2 mol 1-1 0.2 NADH 2.25 mmol 1~4 0.2 ADP 1 5• 0 mmol 1~1 0.1 GDH 0.05 Homogenate sample 0.2 M onitor absorbance a t 340 nm.
2 . 2 . U t i l i s a t i o n o f NADH.
The main o b serv ab le fe a tu re o f the b la n k re a c tio n was the f a l l in absorbance a t 340 nm and i t . was assumed th a t th is was due to the o x id a tio n o f NADH to NAD . I t was f e l t n ecessary to c la r if y the f a te o f th e NADH, the re b ein g the p o s s ib il ity th a t th e NADH was being removed by some re a c tio n o th e r th a n the o x id a tio n to NAD+ . This problem was approached in two ways, namely, th e s p e c tra l changes o ccuring d u rin g th e o x id a tio n o f MADE and also the flu o rim e tric q u a n tita tio n o f NAD+ fo rm atio n .
Normally i t would be expected th a t the o x id a tio n o f NADH (a b so rp tio n maximum 340 nm) to NAD+ (a b so rp tio n maximum 260 nm) would be accompanied by a f a l l in absorbance a t 340 nm and an
in c re a se in absorbance a t 260 nm. If , however, th e o b servable f a l l in absorbance a t 340 nm was r e la te d to the d e s tru c tio n o f NADH ra the r th an i t s o x id a tio n , th en some a lte r n a tiv e s p e c tra l changes m ight o ccu r. S tu d ies o f a b so rp tio n s p e c tra were com plicated by the presence of ADP (a b so rp tio n maximum 260 nm)and to overcome t h i s d i f f i c u l t y th e experim ents were perform ed in the presence o f
-1 *
1 oO mol 1 NaCl to s ta b iliz e the g lu tam ate dehydrogenase. In
c o n trol experim ents u t i l i z i n g commercial L-amino a c id o x id a s e ,it was p o s sib le to dem onstrate th a t the f a l l in absorbance a t 340 nm was p a ra lle le d by an in c re a se in absorbance a t 260 nm (Figure 17* P 9 0 * However, in experim ents u sin g .tis s u e homogenates, due to th e h ig h absorbance produced by th e n atu re o f th e s a m p le ,it was n o t p o s s ib le to dem onstrate th a t the absorbance a t 260 nm ro se s i g n if ic a n tly .
The form ation o f NAD+ d u ring the re a c tio n was however c o n c lu siv e ly dem onstrated by th e use of th e flu o rim e tric method. The NAD c o n te n t o f a liq u o ts tak en from the re a c tio n m ixture were compared w ith th e f a l l in absorbance a t 340 nm o f th e re a c tio n m ix tu re. The r a te o f f a l l in absorbance a t 340 nm expressed as a percen tag e of th e t o t a l f a l l , showed a mean v alu e o f 3«0$,which compared fav o u rably w ith th e mean value fo r the percen tag e r a te o f in c re ase in flu o resc en c e of 2 .8 $ .
91