a Atributos de delegación, obligación y precisión

P ig 6i A b so rp tio n spectrum o f cytochrome £3 5 2 . 5 !<--- M > . o x id is e d ; ( ---A te),a sco rb s t o ­ re due cd ; ( , JL) d i t h i o n i t e - r c d u c e d . 1 cm c u v e t t e contsin ed 0.32 mg

o f h i g h l y p u r i f i e d cytochrome £5 5 2 . 5 » " 50 "W Tris-H C l b u f f e r . pH 7.3, in a f i n a l volume o f 1 m l. The a s c o r b a t e - r e d u c t io n and d i t h i o n i t e

redu ction were allowed to proceed f o r 13 min and 2 min r e s p e c t i v e l y b e fo r e re c o r d in g the spe ctra .

153

TABI.E64. EFFECT OF AMOUNT ANI) OKOEK OF ADDITION OF CYTOCHROME £5 5 2 . 5

AND CYTOCHROME £5 5, ON TIIE ACTIVITY OF THE THIOSULPHATE: CYTOCHROME c OX 11X3REDUCTASE SYSTEM.

F i r s t a d d it io n Cyt % 5 2 . 5 Cyt % 5 1 (mg) Second A a d d it io n c y t £ 551 c y t £ 552 j (mg) A c t i v i t y

(nmol cyt c reduced - i n " ” (nmol c y t c • - 1 min mg reduced 1) 0.03 0 . 1 2 21.4 70 0.08 0 . 1 2 21.4 58 0.018 0 . 1 2 18 61 0.03 0.17 28 79 0.03 0.08 18 67 0 . 1 2 0.03 16 52 0 . 1 2 0.08 17 46 *

Basic r e a c t i o n mixture co n ta in ed: enzyme A, 0.1 mg; enzyme B( 0.0 8 mg; 2-

S^O^ , 2 umol; horse heart cytochrome c , 80 nmol; T r is-H C l b u f f e r , pH 7.3 45 umol and cytochrome £ 552 5 and cytochrome £551 a* i n d ic a t e d ^ in * f i n a l volume o f 1 ml. Cytochrome S j j j o r cytochrome £ 5 5 2 5 wa* added l a s t as in d ic a t e d , Reduction o f horse heart cytochrome £ at 550 nm was recorded s p e c t r o p h o t o m e t r ic a lly a t 30°C as d e s crib e d in Methods.

p r e v e n t e d t h e good i n t e r a c t i o n between cytochrom e 5 and l ' ,c two enzyme: and caused t h e l o w e r a c t i v i t y i f cy to ch ro m e was added b e f o r e cytochrom e T a b l c 6£ a l s o d e m o n s tra te s t h a t the amount o f cytoch rome 5 t o Promotc t *,e a c t i v i t y o f the m u lti-e n z y m e system was about S t im e s l e s s than t h a t o f c ytoch rom e £ 55^-

Although the s i n g l e a d d it io n o f cytochrome

£552 5 or cyCochrome

£551 with enzyme A and enzyme B c a t a ly s e d the re d u ction o f mammalian cytochrome by t h io s u lp h a t e , the o x i d a t i o n o f t h io s u lp h a te to sulphate was complete o n ly in the presence o f a l l fo u r compounds (T a b lefi5 ) apart from the presence o f cytochrome oxida se and horse heart cytochromes. The complete o x i d a t i o n o f t h io s u lp h a te was a l s o confirmed by experimen ts w ith ^ S - l a b e l l e d th io su lp h a te to measure the fo rm ation o f su lph ate. The r e a c t io n mixture w ithout cytochrome

—552 j gave no r e a c t i o n at a l l . However, the r e a c t io n mix ture without cytochrome C j ^ d id show v e r y low r a t e o f oxygen uptake and an incomplete

2-

o x i d a t i o n o f thio su lp h a te w ith a r a t i o o f Oj to about l . This f i n d i n g was i n t e r e s t i n g in the sense that i t showed a p a r t i a l r e a c t i o n , but the low o x i d a t i o n r a t e d id not en able us to sep ara te and i d e n t i f y the in te rm e d ia te s o r th e p r o d u c t (s ) o f the p a r t i a l r e a c t i o n using ^ S - l a b e l l e d t h io s u lp h a te .

The d i f f e r e n t o b s e r v a tio n s ob tain ed with the s p c c tro p h o to m e tric method and w ith oxygen e l e c t r o d e methods were probably due to ( a ) the former method b e in g much more s e n s i t i v e ( o v e r 100 times in the terms o f number o f e l e c t r o n s tra n s p o rt e d f o r a response o f one d i v i s i o n on the ch a rt p a p e r ) than the l a t t e r ; ( b ) the r e a c t io n c o n d it io n s were not the same: f o r example, the i n t e n s i v e s t i r r i n g used in the oxygen e l e c t r o d e might a f f e c t o r d isr u p t i n t e r a c t i o n s between components o f the cosq>lex which were e s s e n t i a l t o the

r , , c t ^o n * A t y p i c a l p r o f i l e o f oxygen e l e c t r o d e measurement was shown on F i g . b .7

T a b l e d shows that cytochrosw

£.552

5 an<*

cytochrome

£551 a» w e ll as cytochrome £j j q ( b a s i c ) were g r a d u a lly reduced by t h io s u lp h a te in the presence o f ensysn A and enzyme H. The redu ction o f cytochroaic c ^ 0 ( b a s i c ) was

TABLETS. OXID ATIO N OK THIOSULPHATE BY THE PUR 1K I ED COMPONENTS R ea ction m ixtu re Rate o f 02 uptake (nmol Oj m in ~l) 2

-

S 2O3 added (nmol) 0 2 uptake (nmol) Complete 22

100

204 —cytochrome £ 552 5 no r e a c tio n —cytochrome £ j5j 1 .6

100

88 -Enzyme A no r e a c tio n ■ Enzyme B no r e a c tio n

The experim ent was done in an oxygen e le c t r o d e as d escrib ed b e fo r e £ 3 3 * Standard re a c tio n m ixtu re co n ta in ed : enzyme A , 0.4 mg; enzyme B, 0 . 2 mg; cytochrome

5*

0 ,0 9 m&: cytochrome

£351•

0 .2 3 mg; horse h eart

cytochrome

£>

0 . 5 mg; bovine h eart cytochrome o x id a s e ,S u nits, and T ris-H C l b u ffe r SO mM, pH 7 . 3 , to a f i n a l volume o f 1 m l. Na^SgO^ (100 nmol

p r e c is e ly ) was added to s t a r t the r e a c tio n . A l l o f the components were h ig h ly p u r ifie d , and there was no s u lp h ite:cy to ch ro m e £ o xid o red u ctase a c t i v i t y a t a l l in th e enzyme B and the cytochrom e fr a c t io n s . A l l o f the fig u r e s w ere means o f th ree e x p e ris w n to l r e s u lt s . A d e t a ile d d e s c r ip t io n o f the involvem ent o f s u lp h ite:cy to ch ro m e £ o x id o red u cta se in the th io s u lp h a te o x id a tio n is in p r e p a ra tio n (Lu and K e lly , u n p u b lish ed ).

157

Kit;. (>. 7 A t y p i c a l p r o f i l e o f the oxygen e l e c t r o d e exp erim en t t o determ ine the s t o i c h e i o m e t r y o f t h i o s u l p h a t e o x i d a t i o n by the

h i g h l y p u r i f i e d components. The e x p e r i m e n t a l c o n d i t i o n s were as d e s c r i b e d in the le g e n d to T a b l e 6 . 5 e x c e p t th at the f i n a l volume was 0.9 5 ml. Thus, 205 nmol ( 2 . 6 x 83 x 0 . 9 5 ) o f 0 2 was consumed f o r t h e o x i d a t i o n o f 100 nmol o f t h i o s u l p h a t e .

I* t

com plete in th e sense that the supplementary a d d itio n o f d i t h i o n i t e d id not in c re a s e the absorbance any more, whereas re d u c tio n s o f cytochrome Cj j j j and cytochrome £551 were o n ly p a r t i a l , about 60Z and 70Z o f f u l l y reduced form r e s p e c t iv e ly . Cytochrome £j5j was n e a r ly f u l l y reduced by supplementary a d d itio n o f s u lp h it e : cytochrome c o x id o re d u c ta s e . The reduced cytochrome £5 5j was s lo w ly and p a r t i a l l y r e - o x id is e d . On th e o th e r hand adding s u lp h ite and s u lp h ite :c y to c h ro o w £ o x id o re d u c ta s e d id not e f f e c t the fu r t h e r re d u c tio n o f cytochrome «j j j j , which was c o m p le te ly reduced by d i t h i o n i t e . Horse h ea rt cytochrome £ a ls o was f u l l y redu ced by th io su lp h a te in the presence o f enzyme A and enzyme B alth ou gh the r a t e was v e r y slow .

These o b s e r v a tio n s were in good agreement w ith the f in d in g that cytochrome £ ^ ? j and cytochrome were p a r t i a l l y reduced by a sco rb a te and f u l l y reduced

by d it h io n it e ( F i g i > a , ( i ) , which c l a a r ly in d ic a te d th a t th e se two cytochromes c o n ta in two red ox c e n t r e s , a h igh p o t e n t ia l o n e, which was reduced by a sco rb a te and th io s u lp h a te and a low p o t e n t ia l one, whose r e d u c tio n was e f f e c t e d by d it h io n it e o r th io s u lp h a te in the case o f cytochrom e t j j j . These fin d in g s were

confirm ed by ted o x p o t e n t ia l t i t r a t i o n ex p eria w n ts (L u , P o o le and K e l l y , in p r e p a r a t io n ).

6 .3 . 1 1

P u r if ic a t io n o f cytochroaw £ 5 5 0 ( b a s i c ) . Cytochrome ( b a s i c ) was p u r if ie d t o about 8OZ p u r it y by the procedures d e s c rib e d in the M a t e r ia ls and Methods md summarised in T a b l e t s . On the b a s is o f 80Z p u r it y and supposing a y i e l d o f

50Z the cytochroaM comprised about 0 . 3Z p r o t e in o f the crude e x t r a c t . Some p r o p e r t ie s o f cytochrome £ sso ( b a s i c ) . The m o le c u la r w eigh t was determ ined by SDS-gel e le c tr o p h o r e s is to be 15,000. The i s o e l e c t r i c p o in t o f the cytochroam was about (o r higher than ) pH S by the f a c t th a t the band f o r

the cytochrome appeared a t the cop o f the d en a tu rin g g e l a f t e r e le c tr o p h o r e s is a t pH • . $ .

The cytochroaM was complacely reduced by ascorbate as w ell as d ith io n ite

TABLE6.3- P U R IF IC A TIO N OF CYTOCHROME C

55Q(bo»ic) and £55Q(acidic)

P u r if ic a t io n P r o te in P u r ity index*

step (mg) _{<A550 re d /A280 ox*}

(b a s ic ) Crude e x tr a c t 49,100 - Ammonium sulphate f r a c t io n ( A i } Z ) 1 2 ,0 0 0 - DEAE-Sepharose C L-6B (OM N a C l ( l l ) f r a c t i o n ) 234 0.25 Sephadex G-75 55 0.83 —550*, c i d i c * Crude e x tr a c t 39,500 - Aasaonium sulphate fr a c t io n (A65Z) 11,400 - DEAE-Sepharose C L-6B (0.2M N a d ) 660 0.18 Sephadex C-100 125 0.51 Sephadex C-75 SO 0.76 *a ee Ta b le 1

160

The a b s o rp tio n maxima o f the p y r id in e ferrohaemochromogen o f cytochrom e Cjjjj (b a s i c ) were a t 550, 521, 414 ns. On th e b a s is o f the m olecu la r w e ig h t o f the cytochrome and a p u r it y o f 80Z the m i l l i n o l a r e x t in c t io n c o e f f i c i e n t was found t o be 26 cm- 1 ( A ^ ) , which was v e r y c lo s e t o th a t o f mannalian cytochrome c . T h e r e fo r e , the cytochrome a p p a re n tly con ta in ed one haem group p er s o le c u le .

Cytochrome (b a s ic ) was s lo w ly but c o m p le te ly reduced by th io s u lp h a te in th e presen ce o f ensyne A and ensysm B (T a b le6.6) as was h orse h e a rt cytochrom e c. However, the cytochrom e d id n ot a c c e le r a t e th e o x id a t io n o f th io s u lp h a te b y the m ulti-enzym e system swaaured e it h e r s p e c t r o p h o t o m e t r ic a lly o r p o la r o g r a p h ic a lly .

6 .3 . 1 2

P u r i f i c a t i o n o f cytochrosm c ^ Q ( a c i d i c ) . Cytochrome c ^ ( a c i d i c ) was p u r i f i e d

In document La acción de las organizaciones regionales en los casos de crisis democrática de Paraguay 2012 y Brasil 2016 (página 54-60)