Contexto de surgimiento.

350

400

450

500

550

600

Wavelength

K i g . 6 5 < * ) A b s o r p t i o n spectrum o f p y r id in e hacaochronogcn o f cytochrome c5J1 ( --- ) o x i d i s e d , ( ______ d ) d i t h i o n i t c - r c d u c c d . I cm c u v e t t e co n ta in ed 0 . 6 ■* o f p u r i f i e d cyto chrom e c ^ i n 1 mi o f a l k n l i n c - p y r i d in e s o l u t i o n .

Abs

orb

an

ce

(b)

1 4 8

350

400

450

500

550

600

Wavelength

Kig.

6-5

( b ) A b so rp tio n spectrum

o f p y rid in e haeoochronogon o f cytochrome £jj j ( ---- ---** ) o x i d i i e d , ( ______ _ d ) d i t h i o n it e - r e d u c e d . I cm c u v e t t e co nta in ed 0 . 6 mg o f p u r i f i e d cytochrome £ 5Jl in I ml o f a l k a l i n e - p y r i d i n e s o l u t i o n .

r e s u l t s i t cnn be t e n t .it i v c I y c o n c lu d e d tlmt the c y to c h ro m e con t ni ns fo u r to f i v e e - t y p e hnem groups per m o l e c u l e .

•\ 3 .C ~

P I v n lu c end t h e s t n b i l i t y o f cytochrom e The i s o e l e c t r i c p o in t o f

cytochrome Cj j j was found to be pll 5.2 ( - 0 . 3 ) . The slo w movement and the d i f f u s e banding o f the p r o t e in on the 5Z p o ly a c r y la m id e g e l preven ted the p r e c is e d eter m in a tio n . P r e p a r a t i v e i s o e l e c t r i c fo c u s in g on Sephadex IEF w ith Pharmalyce

(pH 2.5 to 5, o r 4 - 6 . 5 ) f a i l e d to p u r i f y the cytochrome c ^ j a l s o due to l i t t l e movement o f the cytochrome on the g e l f o r u n c lea r re a s o n s.

o o

Cytochrome was reasonably s t a b l e when kept i t 4 C o r at -20 C. Thawing and f r e e z i n g th ree times had no d e t e c t a b l e a f f e c t on the a c t i v i t y o f the cytochrome in the t h i o s u l p h a t e - o x i d i z i n g m ulti-enzyme system.

8 . 3 . 7

P u r i f i c a t i o n o f cytochrome £552 5* Cytochrome £ 5 5 2 5 was Purii>-e<* to homogeneity by the procedures d e s c r ib e d in the M a t e r i a l s and Methods. The o v e r a l l

p u r i f i c a t i o n s tep s are sunmarized in Table6.2. Assuming a y i e l d o f 60Z, the cytochrome comprised about 1.5Z o f the t o t a l p r o t e i n o f the crude e x t r a c t . The p u r i f i e d p r o t e i n gave a s i n g l e d i f f u s e band a f t e r SDS-gel e le c t r o p h o r e s i s and there may be a sm all amount ( l e s s than 2Z) o f con ta m in a tin g p r o t e i n s .

P u r i f i c a t i o n o f the cytochrome by p r e p a r a t i v e i s o e l e c t r i c fo cu sin g on Sephadex was unsuccessfu l because the cytochrome moved ex trem ely slo w ly and was, f o r unknown reason s, v i r t u a l l y unfocused on the g e l .

•3. 3. 8

M olecular weight and P2 va lu e o f cytochrosie C j j j j . The sx>lecular weight o f the n a t iv e cytochrome c . . . . was 56,000 ( - 2,0 0 0) as determined by g e l f i l t r a t i o n

( Fij. S.fokJ"552*5

on Sephadex 0-100^ A f t e r SDS-gel e l e c t r o p h o r e s i s a s i n g l e p o ly p e p tid e sp ecies was found, co rrespo nd ing to a subunit a w le c u la r w eigh t o f about 29,000 ( - 2,000). Thus the cytochrome appeared to be a dimer o f subunits o f equal molecular s i t e .

The i s o e l e c t r i c po in t o f cytochrome t j j j j was measured by i s o e l e c t r i c fo cu sin g on po lya cryla m ide g e l and a valu e o f 4 .8 ( - 0 .2 )w a s obtain ed.

130

TAD1.K6.2. PUK I FI CAT ION OK CYTOCHROME

P u r i f i c a t i o n s te p P r o t e i n (mg) * P u r i t y index ( A 552.5 r e d /A280 o x ) Crude e x t r a c t 39,500 - Ammonium s u lp h a t e f r a c t i o n (A65Z) 11,400 - DEAE-Sepharose C L -6B (0.35M NaCl ( I I ) ) 962 0 . 4 5 Sephadex C-100 568 0 .7 3

C h rom a tofocu sin g 378 0 .7 7

Due t o the p r e s e n c e o f n u c l e i c a c i d s and o c h e r c - t y p e cytochrom es d e t e r m i n a t i o n o f t h e p u r i t y in d e x was i m p o s s i b l e b e f o r e the io n exchange c h rom atograp h y.

151 6. 3. 9

S p c c t r . i l p r o p e r t i e s nn«l li.icm c o n te n t o f c y to c h r o m e

y

A b s o r p t i o n maxima o f a s c o r b a t e - o r d i t h i o n i t c - r c d u c c d c y to c h ro m e 5were a t 552.5, 523 and 418 nm and that o f the o x i d i z e d form a t 415 nm as shown in F i g l - £ .

But a scorba te only p a r t i a l l y reduced the cytochrome (about 65Z o f t o t a l l y reduced s t a t e ) whereas d i t h i o n i t e co m ple te ly reduced th e cytochrome. On the b a s is o f a m olecu la r w eigh t o f 56,000 the m i l l i m o l a r e x t i n c t i o n c o e f f i c i e n t o f 28 a t

Aj j j 5-540 an<* ° * ^ a t A552 5 were o btain ed f o r f u l l y reduced cytochrome Cj j j y P y r i d i n e haemochromogen o f d i t h i o n i t e - r e d u c e d cytochrome

C

jjj

j

r e v e a le d a t y p i c a l £ - t y p e a b so rp tio n spectrum with maxima a t 550, 520 and 415 nm. The m i l l i m o l a r e x t i n c t i o n c o e f f i c i e n t o f the band was determined to be 67 cm * , from which a valu e o f 2.3 haem groups per m o lecu le o f cytochrome

C

jjj

^

was e s t im a t e d . The iro n co ntent was found to be 2 .2 Fe per cytochrome c _ . , , . T h e r e fo r e i t appears that the cytochrome c o n t a in s two c -t y p e haem group per m o le c u le .

A cid acetone (0.012N HC1 in a ceto n e) d id n o t e x t r a c t the haem groups from cytochrome £ 3 3 2 S ' w**ich in d ic a t e d that they w ere c o v a l e n t l y attach ed t o the a p o p ro tein .

Cytochrome £ 552 5 uas reasonably s t a b le when kept a t 4°C o r -20°C. Thawing and f r e e z i n g three times had no d e t e c t a b l e a f f e c t on the a c t i v i t y o f the

cytochrome in the t h i o s u l p h a t e - o x i d i z i n g multi-en zym e system.

<7.3.1 0

Stu dies on the involvement o f cytochrome £3 ^ 2 5 an<* cytochrome £3 3^ in thio su lp h a te o x i d a t i o n . Both cytochrome £332 3 o r cytochrome £33^ could e f f e c t the redu ctio n o f mammalian cytochrosw c by thiosu lphate in the presence o f enzyme A and enzysw B as shown p r e v i o u s l y [ i ] . The r a t e o f the r e d u c t io n w ith cytochrome £332 3

as c s t a l y s t showed a p r o g r e s s iv e in crea se w h ile cytochrome £3 3} gave a p r o g r e s s i v e l y d e c re a s in g r a t e , i . c . the ra te reached the h i g h e s t l e v e l immediately a f t e r the s t a r t o f the r e a c t i o n , then slo w ly d e c lin e d . A p o s s ib ly r e l a t e d phenoswnon was shown on Tabletf.4 where adding cytochrome £352 5 i n t o the re a c tio n mixture as the l a s t component govc lower a c t i v i t y than did a ddin g cytochrome

C

jjj l a s t . I t seems that both cytochromes in t e r a c t e d with enzyme A and enzyme B in sosie way and the in t e r a c t i o n w ith cytochrome £551 Bsve lo wer a c t i v i t y , which a l s o

I 32

In document La acción de las organizaciones regionales en los casos de crisis democrática de Paraguay 2012 y Brasil 2016 (página 46-54)