CARLOS R TOBAR

Time of Absorbance Fluorescence

sample a t 340 nm _% 0 0.88 8 5 0.72 30 15 0.48 45 25 0.22 63 35 0 88

Rate of _{0.026 min- ^ 2 .3 min“1} re a c tio n

Rate as a

percentage 3-0 2 .8

of t o t a l change

The absorbance a t 340 nm of a re a c tio n m ixture p rep ared as in d ic a te d below, was m onitored fo r 35 m inutes and the NAD+ content o f samples removed from the re a c tio n m ixture, determ ined u sin g the flu o rim e tric method (Method 5 ).

PROTOCOL

Reagent C o n cen tratio n Volume (cm^) G lycylglycine b u ffe r 0.1 mol 1~1

0 .2 mol I " 1 2.25 mmol 1“ 1 2.05 2 -o x o g lu tara te 0 .2 NADH 0 .2 ADP 15*0 mmol 1“ 1 0.1 GDH 0.05 Homogenate sample 0.2

M onitor absorbance a t 340 nm and remove samples fo r flu o rim e tric assay (Method 5 ).

2 . 3 . G e n e ra tio n o f am m onia.

The fa c t th a t both glutam ate dehydrogenase and o x o g lu ta ra te were re q u ire d f o r th e b lan k re a c tio n suggested th a t, ammonia was b ein g

formed and sub sequ en tly u t i l i z e d by the in d ic a to r enzyme system .

I t was decided th e re fo re to m onitor th e ammonia le v e ls in a re a c tio n m ixture which co n tain ed a l l th e n ecessary components b u t lacked th e in d ic a to r enzyme glutam ate dehydrogenase. The h y p o c h lo rite -

n itro p ru s s id e method was used fo r the d eterm in a tio n o f ammonia (Method 8* p128) Lubochinsky & Z a lta 0 9 5 4 )> b u t i t was very soon re a liz e d th a t g ly c y lg ly c in e a lso re a c te d in a somewhat s im ila r manner to ammonia a n d ,a t the high c o n c e n tra tio n used in the b u ffe r,c o m p le te ly masked any co lo u r developm ent due to th e presence o f ammonia.

P ro te in also acted in a s im ila r manner and a t a c o n c e n tra tio n o f

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5.0 g 1 masked any colour developm ent which may have been due to low c o n c e n tra tio n s o f ammonia.

Bearing in mind th e r e s t r i c t i o n s d iscu ssed above, a s e r ie s of experim ents were u n d ertak en in which minimal amounts o f homogenate and g ly c y lg ly c in e were used in a re a c tio n m ixture based on 0»1 mol 1 phosphate b u ffe r pH 7 .5 , the amount o f ammonia formed b e in g

o -

m onitored over^l hour p e rio d . Using the in fo rm atio n gained in the previous s tu d ie s on th e e f f e c t o f g ly c y lg ly c in e c o n c e n tra tio n s on th e r a te o f the b lan k re a c tio n , a f i n a l c o n c e n tra tio n of 0.005 mol 1 was s e le c te d . Reactio n m ixtures were p rep ared which co n tain ed a l l the n ecessary components and to th e t e s t , b o th homogenate and .g ly cylg ly cine were added, w h ils t th e c o n tro ls contained e ith e r homogenate o r g ly c y lg ly c in e . • 1 1

Only the t e s t m ixture showed a continuous f a l l in absorbance a t 340 nm which, due to th e sm all q u a n tity o f sample used, amounted to

-1

a r a te o f 0.008 min . Samples were tak en from a l l th re e m ix tu res a t 0, 30 and 60 m inutes fo r ammonia d eterm in a tio n s and th e in te n s ity o f th e r e s u ltin g colour was measured in term s of absorbance a t 560 nm.

Both c o n trols showed e le v a te d but r e la tiv e ly c o n sta n t colour

developm ent over the one hour p e rio d ,w h ils t the t e s t m ixture showed a g rad u al in c re a se in absorbance due to g e n e ra tio n of ammonia

(Table 23. p 9 4 ). Although th e a c tu a l amount o f ammonia g en erated could not be a c c u ra te ly c a lc u la te d , due to the in te rfe re n c e e f f e c t of

Blank reaction - am m onia