Generación Del Modelo Computacional Y Asignación De Parámetros En

B u tan o l extraction of p erip h eral photosystem -I polypeptides was c arried o u t by a modified version of th e m ethod of O h-oka et al. (1987) d etailed in section 2.3.

PSI-C

PSI-A

PSI-B

LHC-

FNR

t PSI-D

66 36 2u 20 1C MW (kDa) F ig u re 3.14

P olyp ep tid e Com position o f Low M olecular Mass P olyp ep tid es R eleased from Triton Isolated Photosystem -I F ollow in g n-butanol Treatm ent.

L aser densitom etric trace of th e polypeptides released from th e T rito n X-100 isolated photosystem -I by butanol extraction (M ethod section 2.3). The released proteins were concentrated over a n Amicon YM2 m em b ran e u n d e r Argon. SDS-PAGE polypeptide sep ara tio n w as perform ed w ith an 8-25% polyacrylam ide g rad ie n t u sin g th e P h a rm ac ia P h astG el system .

T rito n X-100 photosystem -I particles purified on a hyd ro x y ap atite colum n w ere used for th e extractions in order to re stric t th e polypeptides ex tracted to those show n to have an involvem ent in events a t th e acceptor side of photosystem -I.

Following Ai-butanol extraction the Fe-S^m holoprotein w as contained in th e yellow-brown aqueous phase b u t it was fairly heavily co n tam in ated w ith h ig h er m olecular w eight polypeptides. Figure 3.14 shows th e densitom etric trace of th e protein content in th e Ai-butanol ex tracted m a te ria l following electrophoresis on a 8-25% SDS-gel. I t contained a ran g e of polypeptides from 66 kD a to 9 kD a including a sm all proportion of PSI-A an d PSI-B, bound FNR and unpigm ented LHC-I.

W estern blot an d SDS-PAGE analysis indicated th e presence of a com paratively high proportion of PSI-C, PSI-D a n d PSI-E. The identification of the 17 kD a band as PSI-E was based principally on its electrophoretic properties and the knowledge th a t PSI-F (w hich also h as a n a p p a re n t m olecular w eight of approxim ately 17 kDa) will n o t p a rtitio n in to th e aqueous p hase during solvent extraction (Oh-oka et oZ., 1988a). PSI-L, a n o th e r possible contam inant, is hydrophobic an d th erefo re unlikely to be b u tan o l extractable. It h ad also been estab lish ed th a t m ost of th e P S I-F a n d PSI-L w as removed from photosystem -I by 0.5% T rito n X-100 w ashing, very low levels of contam ination by th ese polypeptides would th erefo re be expected.

O h-oka et al. (1987) reported th a t th e ionic stre n g th of th e photosystem -I buffer solution prior to ^-butanol ex tractio n effected th e

relativ e am o u n ts of higher m olecular w eight polypeptide co n tam in atio n following solvent partitioning. In the experim ents p resen ted here v a ria tio n of th e ionic stre n g th betw een 25 and 300mM NaCl m ade little difference to th e p u rity of th e end product.

T he in te n tio n du rin g th is work was to d em o n strate th e necessity of Fe-S^/B ill forw ard electron tran sfe r to NADP^ an d to in v estig ate th e role of o th er p erip h eral acceptor side polypeptides in th e m ediation of those events. T his req u ired fu rth e r purification of th e -butanol e x tra c t in order to clearly define th e respective functions of PSI-C, PSI-D a n d PSI-E. P u rificatio n w as achieved by a num ber of chrom atographic procedures each of w hich will be discussed in tu rn .

Ion exchange chrom atography (Preparation 1)

F igure 3.15 shows a densitom etric scan of th e polypeptides obtained a fte r DEAE Fractogel ion exchange chrom atography of th e n-butanol ex tracted m aterial. The co n stitu en t polypeptides were resolved by 8-25% SDS-PAGE.

Ion exchange chrom atography of the n-butanol ex tracted m ateria l on DEAE fractogel served two m ain purposes:

1.) I t lowered th e relative concentration of th e PSI-A, PSI-B, FN R a n d LHC-I w ith a concom itant concentration of PSI-C, D a n d E.

to th e colum n rem oved th e Ai-butanol in th e aqueous solution. Rem oval of Ai-hutanol w as essen tial as it acted as a h a rrie r to reco n stitu tio n of th e extrinsic polypeptides to th e photosystem -I core complex.

T he d isadvantage of ion exchange chrom atography w as th e loss of valuable polypeptides d uring the w ashing procedure. U p to 50% of th e ex tracted m ateria l was lost in the w ashings which included h ig h levels of PSI-C, D a n d E. T here also rem ained a low level of 22-28 a n d 35 kD a polypeptide contam ination in th e eluted end product. To he c e rta in th a t th ese played no role in reconstitution or h ad an involvem ent in cofactor b inding d u rin g NADP"^ photo-reduction they h ad to he rem oved.

Sephacryl S-200 HR Gel filtration (Preparation 2)

The 7%-butanol extracted m aterial w as passed down a n oxygen free 40 cm X 2.2 cm sephacryl S-200 HR colum n an d eluted over a 1-2 ho u r tim e period. T his step w as introduced to free solvent e x tracted m a te ria l of ^-butanol an d to remove the high m olecular w eight co n ta m in a n ts w hilst m ain ta in in g th e EPR characteristics of th e iro n -su lp h u r redox cen tres of PSI-C. The m aterial eluted in two fractions m easu red hy th e absorbance a t 280 n m (fig. 3.16). The first fraction (fraction 1), w hich w as d a rk brow n in colour, form ed 60-70% of th e total eluted m aterial.

I t p assed down th e column a t a ra te indicative of a high m olecular w eight polypeptide or m ulti-protein unit. The second fraction form ed a diffuse h a n d an d w as yellow in colouration.

P S I-C

PSI-D

PSI-E

LHC-I

PSI-A

PSI-B

FNR

66 36 MW (kDa) n T ' 2U 20 F ig u re 3.15

P olyp ep tid e Com position of n-butanol E xtracted M aterial F ollow in g Purification on a DEAE Fractogel Column.

L aser densitom etric trace of polypeptides a fte r in itia l purification on DEAE fractogel by th e m ethod in section 2.3. The elu ted m a te ria l w as c o n cen trated over a n Amicon YM2 m em brane u n d e r Argon. SDS-PAGE polypeptide sep aratio n w as perform ed w ith a n 8-25% polyacrylam ide g ra d ie n t u sin g th e Pheirmacia PhastG el system .

B c o 00 <N Fi 30 60 90 120 150

Elution Time (m inutes)

F ig u re 3.16

E lution P rofile o f Butanol Extracted M aterial P assed Through a S ephacryl S-200 HR Column.

The bu tan o l extracted m aterial w as concentrated over a n Amicon YM2 m em brane u n d e r Argon. It w as th e n size fractio n ated by p assage th ro u g h a 2.6 X 40 cm S-200 H R gel filtratio n colum n in 20mM Tris-H C l pH 8.0, 5mM dithiothreitol and lOOmM N aC l an d elu ted over a 1-2 h o u r tim e period. E lu ted m aterial w as m onitored u sing a 280 n m single p a th

T horough degassing of both th e column packing m ateria l a n d ru n n in g buffers an d addition of dithiothreitol a t all stag es of th e purification procedure w as essential. F ailu re to carry out th ese m easu res re su lte d in th e g rad u al disappearance of th e yellow colouration in p ro tein fraction 2. E P R an aly sis indicated th a t the loss of colouration w as caused by d eg rad atio n of th e iron-sulphur centres of PSI-C alth o u g h th e binding p ro tein its e lf rem ained intact.

SDS-PAGE an d low tem p eratu re E PR spectroscopic an aly sis w as carried out on both fractions. SDS-PAGE revealed t h a t th e high m olecular w eight fraction 1 was composed of all the 7%-butanol ex tracted polypeptides (PSI-A, B, C, D, E, FNR and LHC-I) which, despite th e large ran g e in m olecular w eights, co-m igrated down th e column.

F ractio n 2 contained PSI-C, D and E which, due to th e lack of resolution of th e column m edia, eluted in one diffuse b an d (fig. 3.17). E PR sp ectra an d w estern blot analysis confirmed the presence of th e iron- su lp h u r holoprotein (PSI-C) in both fractions 1 and 2 b u t in fraction 1 th e low te m p e ra tu re E PR spectrum was altered w ith principle g v alu es of 2.10, 2.08, 2.01, 1.985 an d 1.917 (fig. 3.18). The iro n -su lp h u r cen tres in th is fraction w ere unaffected by oxygen.

C lum ping of th e proteins in the m an n e r observed in fraction 1 w as very costly in term s of polypeptide loss. In order to retriev e some of th ese p ro tein s various a tte m p ts were m ade to solubilise fraction 1. N ativ e page gel electrophoresis indicated th a t tre a tm e n t of th e ^i-butanol e x tra c t w ith 7 M u re a for 30 m in u tes resu lted in th e release of th e co n stitu en t fraction

PSI-C

PSI-E

PSI-D

1 — I---1--- T 29 2L 20 U 66 MW (kDa) F ig u re 3.17

P olyp ep tid e C om position o f Preparation 2 (S-200 HR) F raction 2.

L aser densitom etric trace of th e polypeptides contained in th e second fraction elu ted from th e S-200 HR gel filtratio n colum n. T he colum n w as ru n by th e m ethod in section 2.3. The elu ted m a te ria l w as c o n cen trated over a n Amicon YM2 m em brane u n d e r Argon.

g 2 . 1 0 2 . 0 0 1 . 9 0 I___ 3 0 2 — 1____ 3 3 0 3 5 7 F i e l d mT F ig u re 3.18

The EPR Spectra o f Fractions 1 and 2 Eluted from th e Sephacryl S-200 HR Column.

T he p ro tein m ateria l in 20mM Tris-H Cl pH 8.0, 5mM D ithiothreitol a n d lOOmM NaCl was reduced w ith 0.2% sodium d ith io n ite pH 9.0 for 5 m in u tes, before freezing u n d e r liquid nitrogen.

1.) F ractio n 1. 2.) F ractio n 2.

E P R conditions: microwave power 10 mW., te m p e ra tu re 15K, m odu latio n w idth 1 mT. in stru m e n t gain 250.

PSI-C

G50/S-200 HR

PSI-D

30

2b 10 S e p a ra tio n (mm) F ig u re 3.19

a.) Native-PAGE Analysis o f n-butanol Extracted M aterial.

L aser densitom etric trace of th e polypeptides rele ased from th e T rito n X-100 isolated photosystem -I by butanol ex tractio n (M ethod section 2.3). The released proteins were concentrated over a n Amicon YM2 m em brane u n d e r Argon. Polypeptide sep aratio n w as perform ed by native-

PSI c

PSI-E

PSI-D BREAKDOWN

PRODUCTS

PSI-D

0 10 20 30 S e p a ra tio n (mm) F ig u re 3.19

b.) Native-PAGE Analysis o f it-butanol Extracted P olyp ep tid es Treated w ith 7 M Urea.

L aser densitom etric trace of the polypeptides released from th e T rito n X-100 isolated photosystem -I by butanol extraction (M ethod section 2.3). The released proteins were concentrated over a n Amicon YM2 m em b ran e u n d e r Argon th e n incubate in 7M u re a for 30 m in u te s a t room tem p era tu re . Polypeptide separation w as perform ed by native-PA G E u sin g th e P h a rm ac ia P hastG el system .

PSI-D BREAKDOWN PRODUCTS

PSI-D

20 1UT

MW (kDa)

F ig u re 3.20

a.) W estern Blot A nalysis o f n-butanol Extracted P olyp ep tid es T reated w ith 7 M Urea Labelled With A ntibodies R aised A gainst PSI-D.

The n-butanol extracted m aterial w as tre a te d w ith a ran g e of U rea concentrations in order to disperse th e co n stitu en t polypeptides of fraction 1 a n d increase th e yield of proteins following gel filtration. A fter 7M u rea tre a tm e n t th e polypeptides were size fractionated on a Sephacryl S-200 HR. T he re s u lta n t fraction 2 resolved by 15% SDS-PAGE (m ethod section 2.7) an d labelled w ith antibodies raised a g ain st PSI-D (m ethods section

PSI-C

" I

29 2U 20 U MW (kDa)

Fififure 3.20

b.) W estern Blot Analysis o f n-butanol Extracted P olyp ep tid es T reated w ith 7 M Urea Labelled With A ntibodies R aised A gainst PSI-C.

The n-butanol extracted m aterial was tre a te d w ith a ran g e of U rea concentrations in order to disperse the co n stitu en t polypeptides of fraction 1 a n d in crease th e yield of proteins following gel filtration. A fter 7M u re a tre a tm e n t th e polypeptides were size fractionated on a Sephacryl S-200 HR. T he re s u lta n t fraction 2 resolved by 15% SDS-PAGE (m ethod section 2.7) a n d labelled w ith antibodies raised a g a in st PSI-C (m ethods section

1 polypeptides (fig. 3.19^^). However, a re p e a t of th ese ex p erim en tal conditions using pre-equilibrated gel filtration colum ns to isolate individual fraction 1 polypeptides did n o t significantly disperse fraction 1 or increase th e yield of PSI-C, D or E. In fact, the resu ltin g low m olecular w eight fraction (gel filtratio n fraction 2) w as g reatly depleted in th e b an d a ttrib u te d to PSI-D w ith concom itant increase in th e n u m b er of low er m olecular w eight bands. W estern blot analysis revealed th a t th e disap p earan ce of PSI-D was caused by th e sam e p ro tein d eg rad atio n observed in th e YMIOO filtrate following u rea tre a tm e n t of photosystem -I (fig. 3.20a). D espite th e loss of proteins in fraction I it w as decided to carry o u t a rap id isolation an d purification of strom ally o rien tated polypeptides from fraction 2 in a n a tte m p t to avoid PSI-D degradation.

Sephadex G 50 Gel F iltration (Preparation 3)

Sephadex G 50 gel filtratio n was utilised to gain a g re a te r resolution in order to se p ara te the lower m olecular w eight polypeptides. A Sephadex G 50 gel filtratio n colunrn (100 cm X 1.6 mm) w as degassed an d eq u ilib rated w ith Tris-HCl, 5mM d ithiothreitol, lOOmM N aC l pH 8.1. I t w as loaded w ith concentrated n-butanol e x tra ct an d developed over a 24-48 h o u r period. The polypeptides eluted in 4 fractions as m ea su re d by 280 n m absorbance (fig. 3.21). The peak of each fraction w as collected u n d e r argon an d stored in liquid nitrogen. The polypeptide co n ten t of each fraction w as assayed by low tem p era tu re E PR spectroscopy, w e stern blot a n aly sis an d SDS-polyacrylam ide gel electrophoresis:

F ractio n 1 contained a m ass of polypeptides from 8 kD a to 63 kDa. I t h a d E P R an d antibody binding properties identical to S-200 H R fraction 1. F ractio n 2 contained a m ixture of PSI-E an d PSI-D .

In document Diseño y simulación de la cápsula tecnológica para el proyecto Quito Cables (página 87-104)