4384
1-01. so
Binding of Sodium Deoxycholate by Cytochrome
c1
RECEIVED 1:EERUARY 20, 1938 T h e binding of sodium deoxycholate by the basic protein cytochrome c has been studied by several methods. It was denionstrated by electrophoresis t h a t an equilibrium governs the amount of anion bound by the protein. The molar ratio of protein t o total anion at which the former was converted a t pH 8.55 from a cation t o a non-migrating molecule was 1: 17. Equilibrium dialysis indicates t h a t the binding can be represented by the equation l / R = 1 / 7 2 - ( K / n ) ( l / A )where 1/K and 1/A have the usual significance. n equals the maximum number (Jf bound anions and w a s fouiid to be 12 per protein m o k cule. This value is in close agreement with the number of excess positive charges (+13) calculat,ed t o be present o r i tlie protein mrilecule at pH 8.55. Binding a t p H 11.9 was negligible. .A 315 rnp inaxiin~irriw:1s oh.;erved iii the :iIisorjitioii spectruni of ferrocytochrome c in the absence of sodium dithionite.
T h e purpose of this study was the attempt to determine the nature of the interactioii between cytochrome c and sodium deoxycholate. Bile salts, especially sodium deoxycholate, have proved effective LLS agents promoting the separation of the enzynie cytochrome oxidase from extraneous cellular material. Wainio and his were able t o separate cytochrome oxidase iroin the other cytochromes by the serial addition of sodium deoxycholate. Smith and Stotz,j as well as other investigators before them, have employed sodium cholate. An understanding of the binding of a surface active agent by a pure cytochrome would be useful in the flirther purification of cytochrome oxidase. T h e only recorded studies of anion binding by cytochrome 6 are those of Roeri, Ehrenberg, Paul and TheorelP and C o l ~ i n . ~ The former found t h a t when increasing amounts of chloride ions (or Rr- or Sod=) were added to an acid solution of ferricytochrome c, present mainly as Cyt-2H+, 2 chloride ions were taken up simultaneously to form Cyt-L'H+-%Cl-. The absorption spectrum and the magnetic properties of the protein were affected. Colvin found t h a t the binding of Orange I1 was enhanced by lowering the p H and that the binding of individual anions was affected by previously bound anions. The binding curve is sigmoid in shape.
Methods All studies were conducted with cytochrome c prepared frcini beef heart by the method of Keilin and €Tartree* as further purified by the method of Illargoliash.g The purity o f the resulting cytochrome c wiis measured b y its iron content (0.42--0.474,&), nitrogen content (1-1.4-15.1 R j and electrophoretic homogeneity (one peak at pH 8.55 and P/2 o f 0.2). Difco Lahoratories sodium deoxycholate which ass:iycd a t 99.00S; was used. Iron determinations were i m d e by the cu,oi'-dippridyl method of Kleinio and nitrogen t1ttcriniii;itioiis b y the seini-micro Kjeldahl method. Electrophoresis.-L)eterrninatiotl.; were made a t 0.1'10.5' ivith a field current OF 9.5 i~iillinmp.using the Perkiiii'rcsented a t t h e M i n n e a p u l i s l l e e t i n g of the American CheniiII cal Scicicty, September, 19.5.:. Supported i n p a r t by a grant-in-aid from t h e S e w Jersey Heart Asociation, Inc. ) I h m a thesis submitted in partial fulfillment of t h e Ph.D. ee. D e p a r t m e n t Riochemlstry a n d Physiology, Kutgers University. ) \V. V.' \Vainlo. S . J . Cooperstein, S. Kollen a n d H. Eichel, .Siii n t r , 106,471 (lg-17). (-1) B Eichel, W. W.Wainio. P. Person and S. J . Cooperstein, .I. Hiid. C h e n , , 183,89 (1950). ( . 5 ) I, Smith and E S t o t z , zbiii., 209, 819 (1954). , K. C;. Paul a n d H . Theorell, B i o r h r m .
Elmer apparatus, Model 38. 'l'lic cytocliroiiie (' \v:ts tlissol\-ed in the buffers of pH 8.55 ant1 11.Oo to give a final concentration of 5 mg. per nil. The sodium deouycliolatc as added t o the buffer t o give final c o ~ ~ e n t r a t i o nofs froni 0.0 t o 0.089 M. The solutions were dialyzed overnight a t 4' against the buffer unless thc protein solutions only contained sodiurn deoxycholate. SO loss of the protein xvits r detected; probably the coIicentration of salts used herc prevented penetration o f the rnembmne bj- cytocliroiiic 6 . l ' Because of the intense color of cytochr~~riie i', base lines wcrc difficult t o obtain with the scanning tiiechanisrn. Area measurements were limited, therefore, t o three deterniiiiations for a study of the separation of the proteiti-anim conrples. T h e mobility values were consistently reproducible on successive trials even though the time of dialysis v:iried from 16 t o 48 hours. This is good evidetice that equilibriuul had been achieved. Equilibrium Dialysis.-These determinations crnployed the techniques of Klotz, \Talker anti Pivan,'2 arid involved the determination of the distribution of anions between a buffered solution of the anion on one side of a differentially permeable membrane : i d the buffered proteill~~anion solution on tlie other. The cytochrome c soluti,ms (5 iiig. per in].) wcrc n ~ : ~ t lLIP e in buffer containing the desired concentration of sodium deoxycholate whicli ranged from 0.002 to 0.01 ,\I. ,Equilibration a t pH 8.55 and p H 11.90 was carried out in 3 / r " Viskitig cellophane tubing for from 72 t o 96 hours at 1". Controls were run at each concentration to measure the binding of the anion by the cellophane. dgain there 1v:is no detectablc lriss of protein through the membrane. Sodium deoxycholate concentrations mere determined by t h e method of Szalkowski and MaderI3 as modified t o meet our needs. T h e amount of anion bound by the protein was taken as t h e difference between t h e total concentration of sodium deoxycholate on the outside of t h e membrane anti the total concentration on the inside. T h e close agreemelit for bound anion Concentrations with successive trials v a r y i w in dialysis time from 72 to 96 hours a n d longer indicated that equilibrium had been substantiallyattained. 1-olumechanges on t h e inside and consequently on the outside of t h e I T I ~ I I I branes were corrected for by spectrophotometric deteriniri:ition of the concentration changes of cytochrome c., i.?., D,,,,I + 0.174 X lo8 = m~ilescytochriirne c- pcr ml., \rhcr(. D equals optical density. Spectral Analysis.- Tlic absorption spectra of ferroc!.tilcliroine r- sodium deosycholate mirtures were tictcrnlinctl ovcr a pH range from 5.5 t o 10.5. In the first series, sodium hydrosulfite was used as the reductant and the absorption was measured frciin 320 to 625 mp. In t h e second series, hydrogen gas and platinum black were used for reduction, and the range studied extended from 220 to 600 mp. I n each series, the cytochrome c and sodium deox!.cholate concentrations were kept constant. T h e spectr:t of three solutions were determined simultaneously: ( 1 1 ferroq-tochronie c in buffer of t h e desired PH (plus when nqcessary); ( 2 ) ferrocytochronle c in the same buffer plus anion; ( 3 ) ferroc~tochrorr~c 1- i l l 0 . 1 .lI SazHPOr SaHnPOl buffer of pH 7.4. -. __
no,
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IC. llargoliasli, . V n l u ~ r ,170, 1014 (19.72). (IO) .1. K . Klrin, personal communication.
(11) D. Keilin a n d 1 : 1:. FIartrcP R i r i r h r i n . .I , 39, 28!4 I W 5 1 . ( 1 2 ) I. h f . K l o t z , 1 7 11. \\'V;ilkr.r : t i i d K Ti l'iv:%n,T H I S , T < ) I . K N 4 1 . 6 8 , 1486 (194')). ( 1 3 ) C . R. Sznlkowski :111
