-1
L -leu cin e 0.1 mol 1 Method
— —
3
1 . 2.7 cm of th e assay re a g en t was p ip e tte d in to each of 5
two t e s t tu b es la b e lle d TEST and BLANK and 0.2 cm of sample was added to each tu b e .
2 . Afte r in c u b atin g a t 37°C fo r about fiv e m inutes th e re a c tio n 3
was in it i a t e d in the TEST by the a d d itio n of 0.1 cm of the a p p ro p ria te s u b s tr a te .
3. Both tu bes were in cu bated fo r a f u r th e r 60 m in u tes. 3
4 . To th e BLANK, 0.1 cm of th e same s u b s tra te was added and 3
im m ediately 1 .0 cm o f s o lu tio n from each tube was tra n s f e r r e d to
3 _1
a p p ro p ria te ly la b e lle d tu b es each c o n ta in in g 0.1 cm 2 mol 1 HC1. 5. The c o n ten ts were mixed and allow ed to stand a t room
tem perature fo r 15 m inutes.
6. To both tubes 2 .0 cm of the sodium h y d ro x id e/im id azo le reag en t was added and th e tubes heated a t 56°C fo r 15 m inutes in
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the d ark .
7. The in te n s ity of flu o rescen ce (e x c ita tio n 365 nm, em ission 455 nm) was measured a g a in s t th a t o f a stan d ard of NAD+ which had
METHOD 6
P r e p a r a t io n o f t is s u e h o m o g e n a te s.
1• Tissu e samples were e ith e r sto re d on ic e and th e homogenates prepared w ith in s e v e ra l hours or fro z e n im m ediately and s to re d a t -20°C u n t il re q u ire d .
2 0 The tis s u e samples were free d from f a t, washed in ic e -c o ld 1.0 mol 1 KC1 s o lu tio n and chopped up in th re e volumes (w/v) o f
- 1 - 1
ic e -c o ld 1.0 mol 1 KC1 which contained 1 .0 mmol 1 sodium d eo x y ch o late.
3 . The sample was converted to a fin e b r e i by two 30 second tre a tm e n ts w ith the P o ly tro n tis s u e d is in te g r a to r used a t low speed.
In betw een each tre a tm e n t the tis s u e p re p a ra tio n was cooled in ic e . 4. The tis s u e was homogenised w ith two 30 second tre a tm e n ts w ith the P o ly tro n used a t to p speed (14 OOQrpm) and two 30 second tre a tm e n ts by. so n ic a tio n u sin g the MSE U ltra so n ic D is in te g ra to r, tuned to an am plitude o f 8 m icrons. The sample was a g ain cooled betw een each tre a tm e n t.
5. The homogenate was c e n trifu g e d in a c h ille d r o to r a t 50 000 rpm (150 000 x g)
60 The su p ern ata n t s o lu tio n was d e sa lte d by p a ssin g a 2 .5 cm a liq u o t through a pre-packed Sephadex G 25 column PD-10, p re v io u s ly cooled in th e r e f r i g e r a to r .
7* The e lu a te was d ilu te d w ith an eq u al volume o f g ly c e ro l and s to re d a t -20°C u n til re q u ire d .
METHOD 7
r
Determ ination o f the p ro te in co nten t o f tis s u e homogenates
A. Biu re t method
Reagents
1. Biu re t reag en t
1 ,5g copper, su lp h a te and 6 .0 g sodium potassium t a r t r a t e were
3 3
d isso lv e d in approxim ately 500 cm w a ter, 300 cm of s o lu tio n
_ 1
c o n ta in in g 100g 1 sodium hydroxide was added and the m ixture made up to 1 l i t r e .
_1
2 . Standard p ro te in s o lu tio n c o n tain in g 1Og 1 album in. Method
— — . 3 . 3
1. To 0 .5 cm o f sample or stan d ard s o lu tio n , 4 .5 cm of th e
Biu re t reag en t was added and the m ixture allow ed to stan d a t room tem p erature fo r 30 m in u tes.
2 . The absorbance of the r e s u ltin g s o lu tio n was m easured a t 540 nm a g a in s t a re a g e n t b lan k .
B. T yrosine Method (Lowry e t a l 1 951)
Reagents
1 . Alkaline copper reag en t
1 .0 cm o f both 20g 1 copper su lp h ate s o lu tio n and 20g> 1
3
sodium potassium t a r t a r t e s o lu tio n were added to 98 cm o f a 20g 1 - 1
s o lu tio n of sodium carbonate in 0.1 mol 1 sodium hyd ro x ide. 2. Standard p ro te in s o lu tio n
1 .Og 1 albumin in w ater. Method
— — 3 3
1. To 6 .0 cm o f the a lk a lin e copper reag en t, 0.5 cm of th e sample or stan d ard p ro te in s o lu tio n was added and th e m ixture
3
2. To each tube 0.5 cm o f F o lin and C io c a lte u fs re a g e n t was added and th e m ixture allow ed to stand fo r a fu rth e r 30 m in u te s.
3. The absorbance o f the r e s u ltin g s o lu tio n was measured a t 600 run a g a in s t a reag en t b la n k .
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METHOD 8