Name & aduress of candidate ---------------------------J.F. Morrison,

Department of Biochemistry,

University of Oxford,

south Parks Rd.,

2:!!:!23:!2·

lUl:S.II;!J!: £~!UD IES II 1'U

:nu:cs:JH'IQ;s.:n,xc ~.eli! c!iCL.I

I

(II

(II}

I:UU

(IV)

IV)

i

prop<!X'Ullllii

b!i!C\1

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t

t:r

d .

•

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•

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fa~ wor~ rep~rt~d

tn~ 't<!!n~"' of !U:l .Au&

suhol.lill'.:!hip •

ot

UI on

nn

~· !U.iti10U •• •• •• • • 91 b. • • • • •• • • 96 c. hi.Ul~<lta •• • • • • • • • • d. uiacuca • • • • • • • • •• 11)9

,,. • • •• •• •• • • ua:

l of I

tt• • • •• • • •• U4

'" "' :cKper iaentl!IJ. • • • • •• •• us A

~· hi •• • • •• •• • • 120

d. lli~JtWJII)ion •• • • • • • • • • ua <'J. ~"' • • •• c• •• • • • • 135

p.,C b7

.i.• fJQt • • • • • • •• 1}7

b. illiMt<®J. • • •• • • .. .. lU a. •• • • •• • • • • 14; d. Ll 1 aoaali! ior:~ •• • • • • • • • • 169 e. • • • • • • • • • • 119

VI

on ~->''" ~-

£:ht; t •• •• •• • • 180 b. ,;J:pfl!' 1m;!lfj tl111 • • •• • • •• 181 tlli• till • • • • • • • • •• 181 d. D1lll<l •• • • •• • • • • 191 tf• • • • • • • • • •• 196

• • • • •• • • •• •• 197

'l!hrot~gb:!Ht'ti tllh! di.8i!f$fi;"'U!:Ul•

"-O{ltl na~><il ~"' r<~ f<~~l"f!!Ht flo i6ili: iioidih

in th!!ll ord~?:r

nue 11:1 !;hili)

~ "' l'l:llii • it ~;Ul

axplilri11leniiiil <'hlllil i p !IN

xu.

to

it;

IN (lliill!Uil

IJiiHiHY'J,<Jti!Jl,ii

ill 1;1;1!!

in lilO!ilUI

dlilta:rmine

•

101

O~idation vf o1~r1~ &Oid

l;; ;<~ 1; 111 !HI l" li!l cHii

.....

ii.U t Iii 0 'i,'

l'hkl'

(l.9lll

0.11 lli!l<' !ill tr

tll0

•

li9l0 l

""" "'"'"" 1\iliU!

uv~:r

ox i 1\11!18

2

•

in t olag id11.tion

!on 1#~8 bi

" thllt 0·5 uf " 'i'lilii F¥ll:ll!!!

the (.lei of

• i~oo:Ur ac \'!! !:&13 -tdl l';t(,l •

~.

I

to

t;~} iiitO" -ic Ill <I •

•

• bo.;;itr -

(

h l.lili~~;~,O•Jllit -

•

hl

•

Dtc\Hl ,.

Jt

lit (j.()t)

•

•

. •

a••• ~t ule-aaoult -1/ ik~h.:t:r £$,~ lt

CW!H'$ to i

t

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5

J

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).nd ivi

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to liillO'I!i

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rttt io t two

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)

1

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on one f

i

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ta.>u11on. 11<\.ll'lt or i<lt ii>l• { t - -f;~l~ 1t!fl

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10

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c 1 ic lM.l

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1.!1 > '

tr

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2+ cu. •

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15

7•4. il'il&lil '1•9·

fo:r

•

!

111'1<1; l ltiV~U $

~ f,Huon { i trio

LtC ibi tnti in a it ivlll •

Oohoa !1948) ~ae to ~o~u~ntrat~ th~ en~vme

~iilll>tlllillll tionatlcn, bu.t t:Uiitr .. lil11'tlll no

""'j'·"" .. "' ••ure 111t

,,J.e~:~ trophor~?~t

ptll" ltV or 'l'lle

1f':l.o """Uv1t1

if¥ 1t.

17

by ttli! Ut1• be • i~UllnlttOn l !ot~.nd thht g: l2fCIU' ul c:r

• latt & 1Dii>i:lll ( 1949) repo:r tn.,t

sl.¥ce~~rol llrll'lil 'Iii

~lt'~Pilll'<;.tlonlil •

c"t<'blli<Miltl

( I •

ll"ul't be b~:»th otullU 2+ ot te liiX!d o.jlatdne.

195ll

•

in the U~,i~h,.t a f

{ 1950. 1951.).

Ji>Qt

~~l:+- Wf0ll!

18

1

t,

1\lll\lil detl!lrldt~cll ..ocord to ,:~~>ll.lli;U" (194 7 J b7 lllll>liUliU'••

ml\!rJt of tll(i al:iaorpt ion 1u 11lJl4:H:trl.lr~>otomot!!r

•

•

with cold w~>ta;r to lC lfl.i. ••

11:12+ WliU!I ' :It lo·4 M t

(liiJitllt"<l Vl'$111 tMn r><'l' ~<tr<•li

u.dng a ~lalllil ;;~ loct:rode

for l h:r. l.lc.f,)r~;~ thill

fin til <JOilCI1l!l t:rlit101'.1 of ~·

llllil VICiil 10•" II!.

Iilith !I to 1·4

20

~~r<~ • tJ •5 ts~l. a solution conh!n1~ ~dl\illl

c!ll>•t,M,H>ni U.u ~;<t '1•4 '1!1~1'1 t.nd thll -tlfll!t t Ll0'!1lil in It l'i!iktiii:r~biiitll ~J>t ;oo • After aD

o • 5 ml. of 507~ ( w/v) tUollloroac'l!tic u\1 14.

l;ii,f,!J it \ll!l\1!! Ul ot.f t • ;o

• hle t;

Definition of a unit of aconitase activity

h<i!!j$J!lll>lll;

ciB••""'ull U.c -

21

aeid

pif ., ·4 }OQ 1n the

j •

a lm.p. 1t'l wall!

ut ;q~laul't•::IWmlti l '""'l:u.o,.U:i

,,·:l.ttig '"' MUltn'

bl th~

.,.u

to tt10il<~ at Li:!.eimlw:~ !iii 1\\!r oont~&ined. app:ret~i~<ble

llliHNtliH> ot· :!.roth £1(!!V111rth~:~lelill!l, tne lmiJOl'tw:~;;~e of J'o 2+

an<~ ilt"'ith!l ;.4111 3CU V~.ttors ot au~:;n1 t~><Jo wu~~> contil'tiH!>il.

in

tlms.

tr.~ i:l'll.ft~r. '111!:• l

~~ in~etiv~ti~n tn~

•

•

J:llil! in<~rlllillilled <~lliii>1111• ~otiv

ot tn• 1oms

~~2+ i 5 x lO"'' Ill) ~od • followillg

'fij.TiQU~ p<>rioos ot tillli!l in ot

ifl'op..ration.

V&li"OI:Itd.

V>en o btdniii!d wUh

l;)llff!llro at

•

24

0 5 10 IS 20 25 30

TIME OF PRE-INCUBATION IN MINUTES

•

t 1m-~ • 01$- OtLi t -4 X 10•3 ;J;)

• •

• 0 {

"' orwoll: or tne ptl ot tne &,1101tflm betan end ~t't!iir lnoub&­

t 1on shcwll!d thst al th,h<lfl'i< tht'lre fit~~!!! o emall olt~n·i:l.t~ on

pH ill the ablillll!loe ot butter. th~t~ rei!IUH1:1! were r~>prod~te iblt~~o

trei£al"lll.t icm of aca;uit<Ml!e o.y the Wlllithod of lll-lch!iill&ll 6

• An!insen (1949) tor ~tivation of their ~oonitalile

f:rllto'Uo&hlo U wee poll!l!libl~:~ tl:u.t; l>be1r finill prepa.J"ti;t ion

In ordGr to deter~ine whetner or not thie was

lilih)(;IIJJ.!i&{i[a wea .Pri!ilpurflll aooo:rding to the methOd

tliill!tlil $l>fl }6° I ill thlil f>.r~lillllllt WOl'tl: lihll! t~lllh lll!il!%'111 m!Mlfl

Ill.\ ,50° liU:Id tl:!" Mtiv1UtH! wue multipli<td by .1..1), the

:rillS Ul.t!l

Zt ,.

t

J!ABt.~ J.. 00l'll!HU"i~Son :;;f Acooi t!l.ll~1 lictivitiec in t.h~' :':rllll<mue and "4..b!!lilllce of

~e2~ and ~~lltli!in~ ( t to ) . ActiVF&tiou b,V §&2+- llthl

• _soo, I>H 1·4·

t'<ili!:<HtS rr!illilent re!!1tlts 2+

t":r."c t 1 on Jo add it ions Hionll ,~J)e +

l'otal. Spec.i.t'ic l'oh1 if1c 'J:ohl iJni ts &ctivlt; tini ts iVitJ' units

cific aetiv~tted) enzyme ivlt.)l tHin-ii>chvated l

c..x:tract )10.000 10.0 261.000 10.5 278.000 11.) 1·07

,,, pH 5•6 t~th- 13).000 18.9 144.000 26.9 220.500 41.1 !·53 &.bel

J..i.pR 5•6 eth- 102,000 68 100,500 29·3 175.000 51·5 1·'/4 anol ppt.

p.K 6.8 eth- 6).000 132 45.000 45 105,000 107 "·35 arwl ppt.

the l.i<vt Md n<arl•i!WtlVal>1iid fl"e~Jti!Jilii! ah.Otill

cie~roe of aGtiVIll<tion bJ ii'l!IZ+

tiofl PI'OGllld W'lill•

pr>:rlf teat ion of

ft'Olll tl:~t

p rlrU' il11d

U.bd

obtain

•

It dH dllfO

thlll ill

bJ B~:tebal'lM

bill

"

ilOtl'ld tn~•t thlil relative

~hp d itf<~>re raa:r "1!1 tU.s

hli!Olh M.o:reoTor.

4"''"'~1oo 111tope

1Ul.i!.¥illlill

n !If ill " ~!ppnci!ilhiil

~oonit&@~ furtn~r luvolv~d trial ot

f)l'Ooedurel!l•

liliH' UMid ill 4t~

r;:uni.ltl> ct 1ill these procedur111a Otrlll not

l ~13 lll~WJ' brought lil.bc>ut UtUe or no

ill the puritJt

their ~* iii~ ma4e.

illlllt#llltl, but bri!ll:f i'lUiHltiOl'l

find ml'iiU:\04 for Uui

pur llioa.t ion li'I&Ul IU! f!illlCW$;

&rttr~totiol:l ot tis&Wt

lwl!;;~diatd.Y ~,tfhr t!Hl dl!lutl:&. of allillld.• tne

0,., •• ~,, were :r~cved, OJttt iflto i\ltrip!!! fii:IO<lt i il'l!i!h

thio~nlilelll. fr111ed oonneot1ve tiastte i\lna

ill he.

l; hr 0 u,lh 1!1. O•:l!U" Gil

tll:ltH!Il' tt!!il!l.lll OlllltU tione,

l!!lil!'il!

ot :t'rO~t.!lln

tnen

Ill i!;Jl,)t<t li'UIIi

W II>!!' a.>" L v "'"'"'

~>!!!d in

in~ l:il!llndor "'

IHA:ffu (pH 4 •1 I an~ 150 ffil·

r<~d '""~~ {l

• 11tored ~15°.

!lo stnrflil. for

ivi •

•

!ri.lld 1 tlil le

W!&l! Q!Hlt.l" at •1° !!ill'

• <i.l.l ratiOlilll !Wiil:f'e ell-.rr il'ld cut ill

(

lll'llt:! b:rO tO 0° ill 4!1.

with

tliil•/i>r. •

w litlii wlill!ld

•

1

negbe'la!,dl. tl:l~ W'\11 b·~lng l 0 Ill Ill r:· f>'li to

~m~:~.U @ll!Oiillt ot "' lii'fd. t~:~ i}}itat<l lUll!! eentr

~~,t 0 •4 • of t 11itliuh W$i,lii

\'$'~fi:} J uat11ul to pH 6.6 til li!Od

~on i ®II ii~UJ.Ifll<.te fr<M:U<Hliiit.l.On

o.125 m dtrute

r)6•'1 e•/lov llil·

eolatiou llfuli! "

( 6 .e •.

0 ... . A

ott

,.6. ion.

•

itl 11. w""t~r·~»at!t tor 15

u1; ill oo il.lll~b""tn.

,~tbi"'Uol traoU<Hl~~ttiOfl at pH 8.0.

to 25 ~1. lot• waa

• @t 50° and cool~d

'fh<~ ii!ll.p~u·na,tall t 1:UU'! MOl!ld to 0° and l!lt.i:!OOOl Wll.ili

~tdd.<~d to a c.onoon t.r!ll.Ucm ot 0

J.ow<~r<~h to -5 • '' e~:;a.n 3l!l!o w:~ t

t !:ill$ p!~f' ~~ t ..iJ" ill :1. I'll!

prfloip.Ut<h wae

Uon ot !)Oji~. tho temperature tming low1.1rtH'l to ·l2Q.

Juring the f'l'~><l t1 ooatii.Ull at ~:>lii;,,jlln~ p!:l va1u.e111, carl!! 11'/d

from

l ot

ll!iiillO ul W~J ~;;ul;ph~< te

OlltYrllt1on W~S 0.65 •

•

• it 00

"''"or ph<H<Il! fo:rm.

1l. brief /il-ucooot of tne prooedurei! ldlidl were tried

i>itll,nl.t lli!:AOOI'IIUi! <hir:l.ti,"C tb.lll UOIU'IUI c;f tl'H'l l)lUTifill' .. t!Oil

5•6 ana 6.6 co tlld ~ o bt,.1ned.

wHll g~but~ol tor

tr®utmunt did not ul

33

•

tlul dne rl!laot•ii. willii:t th\\'1 oitrat~:~ Plll'lllll!<~~tili•

ill ~~>l:lb 2 h giV>HI Ill IWl$!1llllill'}' Of th\\'1 ,fielb i!itld

t

pr<!~pAr.,~; lo!h

~>'l'tHHllli'<r" br lnge &boat a 24·fold

~OOQi\al!l~t ~ith ~ OV$l"&ll

G~oeral proplll!rtihll! <Jf a~Hw1tt;Us~

lii!U~¥m<!~ in dil~tti cltrat~:~ b~ffl\'lr at 5•1 re ita aotid t;~

fro:..<>r• 1:! I! <!I Wliklll

t h<>!lf lUi. 11; llf!i.l!> not

8.0 k'pt ..

pJI 6 .. !) ililllliiiOtllll!lil

112

ll6

102

• 15

:tn Conca. ( /rlll.l

6.;

6.;;

19 .. 6

8.4

1·0

16.;

u.s

rotilill

l!?;.l

39·3

5·)

2.2

()

0.12

0 .,~1

(J.l9

in p~t~r

10 431.£JOO

)10 •

212

1410 157.000

1)20 135.000

96.000

50,

! aotivi

ll·l

49

96

168

188

tor cottons)

100

49

}6

31

22

12

u:.

MUvtty 11111$

of ;UJOt\1 taee

tion cc ~~o be fr~•~~~drl~d witho~t 10iii8 of activit¥.

bitt. it !!I!Ul l!IO.f~ QOilV~tihHit tO ;(<!lOP it 111 tile frO&IUl

n • l!'l&• <:. oholllliil tt>~:<tl IIU:ll:lfli h gtcbl~ti to neat

U a, t~mpcrat~<r~ of 51° 10 ~>.tt;>ined, a.fter wnioo tnera

5•7 to 8.

::illllctl·oenvut ic .. n .. l.£chl

. , . .... ,.,.3 1l!<~~lil ll.l!lihlir conditions

ot its 1liloolootrio

400

.J ::!: -"' 1- 300 z :::>

> 1-

> 200 i= u < UJ

"' ~ 100 z 0 ~

0 49° 50° 51° 52°

TEMPERATURE

' '

37

53° 54° 55° 56°

•

• •

• /ml.}.

l. t •

38

DESCENDING BOUNDARY ASCENDING BOUNDARY

. ,. •

t •

0.025l.

• .s

ov n lli!il t

!i(Jt .1 """"n with le 'i.+

•

' •

•

d till th~t

moMUtJ

39

tne tot~l prot~in of h~~rt ~noel~.

i.Oh tl>at tll~ holnt~<l not

booU.r iC li\1/Udii! !!I $11COI:Iltli\$<1i t'l"I>OUOI:llil in thill tirOI>illl:lOfil ., -ani! l\llill'!lllllot~~ of -'•<!+ Md ototidne

i

)• Compt~rldi!()ll ~Jf tlll!l Ratl!lil of Oitr io aeid io:rl:t.t~.tion from Isocitric E~c.ld

!!!.,.ois-.aconitic acid by ActiVtJoted e.r!d lbn-activf,ted .i''r11ctiona of ;lconital!e.

.t#.C t

at. eth<•nol ppt.

2nd. ethunol ppt.

~ su.lpl:u.; te ppt.

1libl,ysed i:illlillH:I !! 1 tim -ppt.

&.ft.er heating at sr,;J

w

iVI!t!ld l>.ll': dt>iHll"i

Specific Activities

Non-activated Activated

(a) (b} Ratio !ol

in the tl!l:xtl

{d l

kti vit ies

!it<.J t 1 vatl!li ~ mzyme io non~act hatid ,

Ci.s-ii<con- iso-oit- a:b cis-aeon- i~~;oeit- o:d Qil!!-act:~n i&lCitric n!e acid rio acid ntc add rle ''cid n!o l•oid --:ltcid

1·0 4·35 1.61 '[.2. 1·59 1.6) l. 66

31 17·5 i.'f8 )l 1·58 1.6 l. 8

50 32·5 1·54 ':!> l.'(l. 1·9 1. 7

'12·5 45·5 1.59 151 91 1. ':!> 2.1 l

f.O 4·1 1·49 85 1.67 20 18

6.1 4·2 l.6o 174 113 l-54 26 27

lO!illil

llllli<J no

of let+

• but

of ifeh.

cO!l<tJl<~ttel~ the

.ltlon ot 2:2 1 ~Uv.vt·U,rl

this·

l!<i:!i:iltl.l for th~ pr~6~r•~;e of oth<i;~f' illl~i;:l!lllla in UH1 .final

.. uouit&IM:> pt'l!l~¢~;;.rllt11J!l

42

lt

l

Ill •

•

7 ~ "'" )•\J\J ,i4.

•

u:

•

' t

t

a or eLeutran:. ur

iLl!

(.nz~ent

•

41

i

•

t

i

l<Jd li& c J..c&.al i

" tl.U'~

:r'll

l,~Ilt ti~~\l~l*

or rAo t

t of

'

i

;.fl

t

•

l 1141~

lU);;tl'!f$

2+ :;Lre~ :>n

of

•

©OilJi;JO;!OU il: II ii$1"11

;;t~ &,fl tlel'(J ?.

Z+ £,;n

49

( )

l

•

•

I

•

!;,J

•

:

+

l

it

' •

•

•

~+

n1· or .;:+

•

l

'

•

+

•

, D~t lt l~ notewort t

•

lit ill® ..... .U.'II~lJU,,;j ill!!;

1e2

"' •

•

\

•

•

'tttit:

2+

t

+

54

•

•

55

I 19 ).

20:.

•

••

iV . •

.J3,000 ::; -"' 1-z ::>

I

> .... -2p00 2: .... u < I.U 1/)

~ zr.ooo

8 "(.

-

l llr•

' t ~

D

•

d(/1 2+ •

"'" 1·4 1\i~ 1D

5 ~ lo·4 Iii ~~!:!+

5 J[ lo~4 lit i'~.l:+ + o.ol M 1ne

~ l5*

~~<1+

but l+

ifll 1n tne

• ,,

•

51

•

2<t £iie ttttHb •

•

1n

J( t:

•

it

{l;ft~IHJt q:t +'~®-1nl.1\lbli!,tio~ Xiille en t.l'•e t.cU~·.,til!!

2• :t.,l Jl.3i>I#U1h~e !§' l!'e ~~>lld ,o,y;at:cei,ne

( A00t1J, t!l<IHl lliiiU! '/•4

le2"' Mil lQ•! AI l.1.lf!\lt'ilifll11• 0.2 ml.

o.) • of el8-~con1tlc !i!C -4 X 10"') li.}

llml!l o! !Cti.~t~1Qil

(br.l

0

o.s 1.0

2·0

;.o 4·0 ~).0

6.o

•

t'li~Jt t~<be!ll eon t&.iaU•

( finl>l c!Ult~i!!lntr~<tion

•

l)1th c i tr acid ftJ1/'IfH$fi/ 15 • )

2o5 :5J5 }20

3!0

)20

}18

295 278

• of

60

•

nnl:l£l<~n .for til~

1 lOU Of t~

I

•

•

• lt

• for

«,oti?l!tti<>n of ~>oonlt..,!li\1 bj; till!' udd:1Uon of J¥'41 2+ Uf<d

"'" i>h in<~ . tl} t!l" tnt rr.ll'U W;~~ ~ !i#h!ii'

fLitb t.I:IIW 2+

' ~:; t.) 0\.tl,

2+

• Iti

• •

•

1V1 • 1;

:I: Iii

tll!:l!

0

t

u;·2 i!.

till\ u.

• It

•

• •

700

fil600

~ 12 500

0

~ 400

!::l 1!: 300

0 Cl 200 ~

100

. ;. l?e:l!+

til! lilt

lo-t: Ill

•

10 IS

TIME- MIN. 20 25 30

pg. ot

fi'f!%'(1

·4 1i' 2+ 5 lt 10 . ~ "(I •

• ¥¥1 itl!!2+ ll ,.., b!ii 1. r· "' t e

63

iii

lil.!!I'J'illl!l~l!l~t.

kllt.iVIIUOn fit IIIIOilUIIIIilil! \li:l!il!ll" <Mill"Obi<~ at~d. !!i.illl!:!l"tJbiu

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