LA FORMA VERBAL EN

Blank reaction

Test

reaction

sample

su bs tra te

T i m e

T ypical tr a c e of a sp ec tro p h o to m etric assay u sin g a Unicam SP1800 sp ectro p h o to m eter. The arrow s in d ic a te th e tim e o f a d d itio n of sample and s u b s tr a te .

r a te depending upon th e e x te n t o f endogenous ammonia form ation by th e sam ple. T his re p re se n te d the b la n k re a c tio n which was measured and subsequently su b tra c te d from the t e s t re a c tio n r a t e . The

a p p ro p ria te s u b s tra te was th en added to i n i t i a t e th e re a c tio n and a f t e r a la g p e rio d ,d u rin g which the re a c tio n r a te in c re ase d to a maximum, th e r a te o f re a c tio n held c o n sta n t u n t i l th e c o n c e n tra tio n o f NADH f e l l to a le v e l a t which i t became r a te lim itin g . The re a c tio n r a te d u rin g t h i s lin e a r p a rt of the re a c tio n was m easured a t 340 nm in term s of absorbance change p er m inute and the enzyme a c ti v ity o f th e sample was c a lc u la te d in In te rn a tio n a l U nits p e r l i t r e o f sam ple. (Hethod 4. p122).

This c a lc u la tio n assumed th a t th e only r a te lim itin g fa c to r in th e assay was th e a c tiv ity of th e t e s t enzyme b u t i t was

a p p re c ia te d th a t th e re were o th e r fa c to rs which could become r a te lim itin g under c e r ta in c o n d itio n s . Although ste p s had been tak en to m a in ta in high c o n c e n tra tio n s of s u b s tra te s and in d ic a to r enzyme in o rd e r to e lim in a te or m inim ise th ese e f f e c ts , n e v e rth e le ss the c o n c e n tra tio n o f NADH p a r tic u la r ly had to be held r e l a tiv e ly low because of i t s high absorbtion a t 340 nm. I t was f e l t n ecessary

th e re fo re to t e s t th e v a lid ity o f the e q u atio n over a wide range o f enzyme a c t i v i t i e s and in o rd e r to do t h i s , a s e r ie s o f d ilu tio n s of sam ples w ith h ig h c o n c e n tra tio n s o f each amino acid oxidase were assayed in th e normal way and the m easured ra te o f re a c tio n p lo tte d a g a in st th e percentage c o n c e n tra tio n o f the enzyme in the sample (Figure 9o p 57). The r e s u lts in d ic a te d th a t th e lin e a r re la tio n s h ip betw een the r a te of re a c tio n and enzyme c o n c e n tra tio n h eld tru e fo r b o th assays only up to an enzyme a c ti v ity of about

150 i . u . 1 ( a n absorbance change a t 340 nm o f 0.06 u n its per m in u te ). Enzyme a c t i v i t i e s beyond th is le v e l re s u lte d in an

in c re a s in g d e v ia tio n from the lin e a r r e la tio n s h ip , p a r tic u la r ly fo r the L-amino acid oxidase a ssa y . Using th i s method i t was p o ssib le to d e te c t a c t i v i t i e s as low as 2 .4 u n its 1 w ithout d i f f i c u l t y ; th is b ein g e q u iv a le n t to an absorbance change of 0.001 u n its per m inute, and w ith care i t was p o ssib le to d e te c t a c t i v i t i e s as low as

- 1

1 . 0 i o U o 1

The p re c is io n o f each method was assessed by perform ing te n re p lic a te assay s on sam ples w ith mean a c t i v i t i e s of approxim ately

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Figure 9