Fin a l C o n cen tratio n o f s u b s tra te
( mmol l - ^)
Rate o f re a c tio n ( A E nin) • D-amino acid
oxidase ( D -alanine )
L-amino acid oxidase ( L -leu c in e ) 0.16 0.004 . 0o017 0.33 O0OO8 0o030 O066 0.014 0.045 1 .00 0.020 0.052 1 .66 0.030 0.056 2.50 0.039 0.057 3o33 0.046 O0O56 6.66 0o058 0.041
M e t h o d
M i c h a e l i s
C o n s t a n t (mmol
I"*)
Graphical
S ta tistical
3-3
3
*3 -042.0 - 7
o . 6 ±o -ik
(Table 11. p 53)* B urton (1955)? and R atner (1 955b), quoted values - 1
between 2 - 9 mmol 1 .
For the L-amino acid oxidase, however, the r e s u lts in d ic a te d a s ig n if ic a n t degree of s u b s tra te in h ib itio n a t c o n c e n tra tio n s of
- 1
L -leu cin e above 3*0 mmol 1 , the in h ib itio n becoming p ro g re s s iv ly g re a te r as th e c o n c e n tra tio n in c re a s e d . (Figure 7 . p 52). These r e s u lts were n o t com pletely unexpected as R atner (1955a), had s ta te d th a t c o n ce n tra tio n s o f L -leu c in e above 10 mmol 1 were in h ib ito r y . She also noted th a t ammonium ions in h ib ite d a t th e same c o n c e n tra tio n .
The n o n -in h ib ite d p o rtio n o f th e Lineweaver - Burke p lo t wa's used to c a lc u la te the M ichaelis co n stan t and i t gave a v alue o f 0 .6
— 1
mmol 1 (Table 11. p 53)- R atner (1955a), had quoted a value o f about 1.0 mmol 1 w h ils t Rakano & Danowski (1971) gave a v alu e o f 1 .3 mmol 1 .
- 1
A c o n ce n tra tio n o f D -alanine o f 0 .5 mol 1 was chosen fo r the working s u b s tra te fo r th e D-amino a c id oxidase assay which, when
used as d e sc rib e d in (Method 2 . p119), gave a f i n a l assay c o n c e n tra tio n o f 17 mmol 1 . This c o n c e n tra tio n was approxim ately fiv e tim es
g re a te r th an the Km v alu e fo r th e s u b s tra te and p e rm itte d 84$ o f the th e o r e tic a l maximum v e lo c ity . In se le c tin g a s u ita b le c o n c e n tra tio n o f L -leu cin e fo r the L-amino acid o xid ase assay , not only was the
in h ib ito ry n a tu re o f L -leu c in e to be considered b u t a lso th e f a c t
- 1 •
th a t i t showed only a lim ite d s o lu b ility o f approxim ately 0 .2 mol 1 , Weast (1967)» As in h ib itio n only became ev id en t a t c o n c e n tra tio n s in
- 1
excess of 3-0 mmol 1 i t was decided to use a working s o lu tio n
- 1
c o n tain in g 0.1 mol 1 which, as w e ll as bein g w ith in the s o lu b ility range o f L -leu c in e , a ls o provided as assay c o n c e n tra tio n o f 3*3 mmol 1 and th e o r e tic a lly p e rm itte d 85$ maximum v e lo c ity .
4 . 6 . Assay p ro ced u re.
A ty pic a l tra c e o f an assay of e ith e r D-amino acid o x idase or
L-amino acid oxidase by th e method d escrib ed is shown in F ig u re 8 (p 55)« On a d d itio n o f th e sample to the re a c tio n m ixture from which the s u b s tra te had been om itted, th e re w as.a ra p id f a l l in absorbance a t 340 nm due to th e o x id a tio n o f RADH by ammonia p re s e n t in th e sample The absorbance e ith e r s ta b iliz e d o r continued to f a l l a t a c o n sta n t