2.1.1 Isolation o f the Thylakoid Membrane
Photosystem -I particles were p rep ared from m a rk e t spinach {Spinacea oleracea). The spinach leaves w ere w ashed an d th e larg e le a f stem s a n d ribs removed. The p la n t m aterial w as th e n ground in a W aring b len d e r for 15 seconds in ice-cold 0.33 M sorbitol, 20mM M es-N aOH, 0 .2mM MgClg pH 6.5 (grinding m edium ) w ith additional sodium ascorbate (5mM). T his m ixture was filtered th ro u g h 8 layers of m u slin a n d th e filtra te centrifuged a t 3,000 g for 5 m inutes to obtain a chloroplast pellet. T he p ellet was resuspended in a hypotonic solution of 5mM MgClg for 60 seconds to osm otically shock th e chloroplasts. T his w as im m ediately followed by th e addition of a n equal volume of double concentration g rin d in g m edium . A thylakoid pellet w as obtained by a fu rth e r cen trifu g atio n a t 3,000 g for 20 m inutes. T his w as resu sp en d ed in 20mM M es-N aO H , 25mM NaCl, 5mM MgClg pH 6.3 (resuspending m edium ) and left on ice, in th e dark, for a period of 2 h o u rs to prom ote thylakoid stacking.
2.1.2 Preparation o f Triton X-100 Photosvstem -I P articles
T he m ethod of B erthold, Babcock an d Yocum (1981) w ith m odifications by Ford an d E vans (1983) w as in itially u sed in o rd er to se p a ra te th e photosystem -I from th e photosystem -II.
T rito n X-100 (20% (w/v) stock) and resuspending m ed iu m w ere added to th e stacked thylakoids so th a t th e final d eterg en t a n d chlorophyll concentrations w ere 5% (w/v) an d 2 mg/ml respectively. The m ix tu re w as in v erted twice, left to digest for 25 m inutes th en centrifuged a t 40,000 g for 30 m in u tes to pellet the bulk of photosystem -IL T he re su ltin g s u p e rn a ta n t w as centrifuged a t 145,000 g for 2 hours th ereb y rem oving a n y rem ain in g photosystem -IL
The photosystem -I (TSF-I) su p e rn a ta n t w as raised to 20m M T ris- H Cl pH 8.0 an d lOOmM NaCl th en dialysed overnight in th e d a rk a t 4°C a g a in st lOOmM NaCl and 20mM Tris-HCl pH 8.0 in p re p a ra tio n for fu rth e r purification by column chrom atography.
2.1.3 P u r ific a tio n o f P h otosystem -I by H v d ro x v a p a tite
C hrom atography
Removal of th e lig h t h arv estin g complex, free p igm ents a n d some low m olecular w eight polypeptides was achieved by application of th e TSF- I to a colum n packed w ith hydroxyapatite.
T he colum n w as pre-equilibrated w ith 20mM Tris-H C l p H 8 a n d lOOmM NaCl, a fte r appfication the sam ple was w ashed w ith 20m M Tris- H C l pH 8 , lOOmM NaCl and 0.5% T riton X-100 u n til th e e lu a te r a n clear. I t w as th e n w ashed w ith 3 colum n volumes of 0.1% T rito n X-100, 20mM T ris-H C l an d lOOmM NaCl to rem ove excess d etergent. The photosystem -I, w hich ap p eared as a d a rk green ban d tow ards th e top of th e colum n, w as elu ted from th e colm nn by slowly w ashing w ith 5-15mM KHgPO^ pH 6 .8 ,
lOOmM NaCl and 0.1% Triton X-100.
The elu ted sam ple was raised to 20mM Tris-H Cl pH 8.0 a n d th e n dialysed for 12 hours in the d ark a t 4°C ag ain st 20mM Tris-H C l pH 8 a n d lOOmM NaCl to rem ove the phosphate buffer. F inally it w as co n cen trated in a n Amicon u ltra filtration cell over a YMIOO m em brane a n d stored u n d e r liquid nitrogen.
All of th e steps during th e purification procedure w ere c arried o u t a t 4°C in th e dark.
2.1.4 P reparation o f D igitonin Photosystem -I P articles
T hylakoids were p rep ared by the m ethod in section 2.1.1. D igitonin iso latio n w as carried out largely by the m ethod of B oardm an (1971).
D igitonin (2% w/v stock) and 50mM Tris-HCl, 5mM MgClg an d 2m M ED TA (TEM ) buffer were added to the chloroplasts to a final concentration of 1.0
m g/m l chlorophyll an d 0.5% Digitonin. The m ixture w as well s tirre d a n d left to digest for 30 m inutes on ice in the dark. It w as th e n cen trifu g ed a t 15,800 g for 30 m in u tes to pellet photosystem -II an d th e p ellet discarded. T he s u p e rn a ta n t w as fu rth er centrifuged a t 144,000 g for 90 m in u te s a n d th e p ellet containing the photosystem -I was resuspended in TEM b u ffer a n d stored u n d e r liquid nitrogen u n til required.
2.1.5 P reparation o f the Photosystem -I Core Complex
The Fe-S^/B protein an d other low m olecular w eight p e rip h e ra l polypeptides w ere rem oved from photosystem -I by tre a tm e n t w ith 6 .8M
u rea . 9M u re a in O.IM Tris-HCl pH 8.0 was added dropw ise w ith rap id stirrin g to a suspension of photosystem -I particles, in itial concentration 1.5 m g chlorophyll/m l. The suspension w as diluted w ith oxygen-free Tris-HCl p H 8.0 to 250 pg chlorophyll/ml, 0.1% 2-m ercaptoethanol an d 6.8M u re a a n d stirre d u n d e r argon a t room tem p era tu re in th e d ark. The rem oval of th e Fe-S^/B p ro tein w as m onitored by m easu rin g th e re-reduction kinetics of P700^ by back reaction from electron acceptors following la s e r flash excitation. K inetic m easu rem en ts were m ade u sing th e kinetic spectrophotom eter outlined in section 2.5. W hen th e t^^ for P700^ re red u ctio n h a d changed from 20 m s an d stabilised a t 1 m s (25-30 m in u tes incubation) th e tre a tm e n t w as stopped by d ilu tin g th e reaction m ixture lOX w ith oxygen-free 20mM Tris-HCl pH 8.0. a n d 5mM dithiothreitol. U re a w as rem oved by w ashing th e p rep aratio n over a n u ltra filtra tio n m em b ran e (Amicon YMIOO) or by centrifugation for 2 ho u rs a t 150,000 g a n d resuspension of the resu ltin g pellet in 20mM Tris-HCl pH 8.3 and 0.1% 2-m ercaptoethanol. The p rep aratio n w as finally co n cen trated to approxim ately 1 m g chlorophyll /m l and stored in liquid n itro g en u n til required.