B uffer
pH 7.5 Rate o f oxygen uptake ( p i min” ^)
B arbitone 0.23
T ris 0.24
T ricin e 0.24
G lycylglycine 0.26
PROTOCOL
Compartment Reagent C o n cen tratio n Volume (cm3) Main v e s s e l B uffer pH 7«5- 0.1 mol 1“ 1
C atalase p repn. Enzyme sample L -leu cin e 20 g 1“^ 2 .3 0 .3 0.2 Side arm 0.2
W ell Sodium hydroxide 100 g 0o2 M onitor oxygen uptake fo r 30 m in u tes.
3 .2 . Effe c t of coenzymes.
No au th o r has sug g ested th a t any coenzyme showed an in h ib ito r y e f f e c t on th e L-amino a c id oxidases b u t i t has been re p o rte d by
R atner ( 1955a), th a t th e p ro s th e tic group fo r th e snake venom enzyme was PAD w hils t th a t fo r the r a t tis s u e s enzyme was FMN. W ellner
(1971)>had re p o rte d th a t the PAD p ro s the tic group fo r snake venom
L-amino a c id oxidase was very tig h tly bound to the apoenzyme and suggested th a t in any assa y method th e re was no need fo r a d d itio n a l coenzyme to be in c lu d e d .
For the p re s e n t study i t was n ecessary to a sse ss th e req u irem en ts o f the' enzyme fo r a d d itio n a l coenzymes and a lso to confirm the
re p o rte d lack of in h ib itio n by o th er n u c le o tid e s . To th is end a sample o f the enzyme was d isso lv e d in 0.1 mol 1 phosphate b u f f e r pH 7 .0 and d ia ly se d a g a in s t the b u ffe r fo r 48 h o u rs. Although th e a c ti v ity of the p re p a ra tio n f e l l d u rin g d ia ly s is by approxim ately 40$, p o ssib ly due to d e n a tu ra tio n of the enzyme, no in c re a se in
a c ti v ity could be subsequently dem onstrated by the a d d itio n o f e ith e r PAD o r FMN, nor did the presence of ADP reduce the a c t i v i t y o f th e enzyme p rep aratio n ,,
The r e s u lts o f th e in v e s tig a tio n s d e sc rib e d above a re summarised below.
L-AMINO ACID OXIDASE
A summary of optim al assay c o n d itio n s and in h ib ito r s .
C h a ra c te ris tic Reagent C o n cen tratio n Optimum pH Optimum b u ffe r A c tiv a to r In hib ito r 7 .5 g ly c y lg ly c in e none L -leu cine 0.1 mol l “ 1 -1 3 .0 mmol 1
4 . S pectrophotom etric a ssa y .
4.1 . C on cen tratio n of 2 -o x o g lu ta ra te .
I t was necessary , having e lu c id a te d th e optim al c o n d itio n s fo r each o f the enzymes, to decide on the co n d itio n s fo r th e coupled a ssa y . The n ecessary s u b s tra te s fo r each o f the two assay methods were ev id en t from an exam ination o f th e b a s ic eq u atio n s fo r the re a c tio n s (E quation 1 & 2, p 7 & 12). Both assay methods re q u ire d 2 -o x o g lu ta ra te and NADH as s u b s tra te s fo r the in d ic a to r re a c tio n ' the oxygen re q u ire d fo r the t e s t re a c tio n was a v a ila b le as d isso lv e d oxygen in the s o lu tio n s .
The re p o rte d Km valu e fo r 2 -o x o g lu ta ra te was 0 .7 mmol 1 —1
Barman (l969)^and f in a l c o n c e n tra tio n s of 13-3 mmol 1 had been used p re v io u sly , E l lis & Goldberg (1970). The c o n c e n tra tio n o f NADH used would depend upon th e maximum absorbance which was capable of bein g a c c u ra te ly measured (p 35) .
4 .2 . E ffe c t of coenzymes.
I t was evident from the s tu d ie s on the a c tiv a tio n and
in h ib itio n o f the th re e enzymes th a t th e re would be some in h ib itio n e f f e c t in the coupled assay, namely ADP a c tiv a tin g glutam ate
hydrogenase b u t in h ib itin g D-amino acid oxidase. However th e evidence
- 1 •
was th a t a t the c o n c e n tra tio n s e le c te d , namely 0.5 mmol 1 , and in the presence o f PAD the in h ib ito ry e f f e c t would be n e g lig ib le
(Table 9° p 45)• 4 .3 . Assay pH.
There was some d if f ic u lty over the s e le c tio n o f th e pH a t which the assay s would be perform ed. In o rde r to g a in maximum s e n s i t i v i t y , i t was d e sira b le to s e le c t the optimum pH o f the a p p ro p ria te amino acid oxidase as the working pH b u t,a s n e ith e r of th ese v a lu es were the most a p p ro p ria te fo r the glutam ate dehydrogenase, the e f f e c t
would be to suppress the a c ti v ity o f th a t enzyme. Prom th e s tu d ie s on the e f f e c t of pH on glutam ate dehydrogenase, d escrib ed e a r l i e r
(Figure 2 . p 33°), i t was c a lc u la te d th a t a t pH 7 .5 ,glutam ate dehydrogenase showed approxim ately 91$ o f maximal a c tiv ity , w h ilst a t pH 8 .3 i t showed approxim ately 85$ o f maximal a c t i v i t y .
4 .4 . Amount of glutam ate dehydrogenase.
In o rde r th a t th e in d ic a to r re a c tio n should not be ra te
lim itin g , i t was decided th a t th e amount o f glutam ate dehydrogenase th a t would p erm it a 100 fo ld in c re a se in re a c tio n r a te over th a t a c tu a lly expected, should be u sed . On th e b a s is o f p rev io u s
ex perience w ith k in e tic assays u t i l i z i n g th e o x id a tio n o f NADH, i t was f e l t th a t a r a te of r e a c tio n comparable w ith a f a l l in absorbance o f 0.1 p e r m inute could be reg ard ed as an ab so lu te maximum, E l l i s & Goldberg (1970) o This r e a c tio n ra te would equate w ith the fo rm atio n
-1 3
o f 0.5 x 10 micromole of ammonia in one m inute in th e 3*0 cm of th e assay and on t h i s b a sis th e coupled assa y would re q u ire a
minimum o f 5 u n its o f glutam ate dehydrogenase p e r 3 .0 cm of r e a c tio n m ix tu re. The volume o f glutam ate dehydrogenase su b seq u en tly used in
3
the assay was 0.05 cm which, acco rd in g to th e s p e c ific a tio n s of th e p re p a ra tio n co n tain ed 60 u n its of a c tiv ity , was more th an enough even allo w in g fo r th e e f f e c t of pH on the a c ti v ity o f th e enzyme.
4«5« Amino acid s u b s tr a te s .
In s ta n d a rdiz in g the assay methods, i t was e s s e n tia l to know the c o n c e n tra tio n o f the amino acid s u b s tra te necessary to give zero o rd e r k in e tic s and in o rd e r to a sse ss th is v a l u e ,i t was necessary to
determ ine th e M ich aelis c o n sta n t (Km) fo r each enzyme u sin g th e
s u b s tra te in d ic a te d . A se r ie s of d ilu tio n s of D -alanine and L -leu c in e -1
c o n tain in g .5 - 200 mmol 1 were prep ared from sto ck s o lu tio n s of each amino acid which, when in co rp o rated in to the assay , gave f in a l
—1 — 1
c o n ce n tra tio n s from 0.16 mmol 1 to 6.66 mmol 1 . U sing Method 2 (p 119), the r a te o f re a c tio n produced by a fix e d amount of the
a p p ro p ria te enzyme in the presence of these c o n c e n tra tio n s o f s u b s tra te , was measured in term s of absorbance change a t 340 nm (F igure 7 P 5 2 ).
Using the s t a t i s t i c a l method in d ic a te d (Method 3* p 1 2 l), the Km value fo r D-amino a cid oxidase was c a lc u la te d to be 3*3 mmol 1
R ec ip ro ca l of ve lo ci ty ( A ^3 4Q nm m ‘ ) Fi g u r e 7