ZEIK IS AQL-EOUS DETERGENT SOLUTIONS
175
( 7 1 FOSIIER, J . R . , .ASDF R E W HD, . : J . .\in. Chetii. SOC.67, G87 (1945). (S) O N C L E Y , J . L . : -J. .4111,Chcin. SOC.60, 1115 (1938). (9) ONC'LI~:I.. J . I,,: .41iii. S .Y,A c t i d . Sci. 41, 121 (1911 1 . ( L O ) O N C - L E JY., L . : (Ilieni. Revs. 30, 433 (1042). (11) SHI.TT.LV, J . : Tt,:ins. F:iixtl:iy Hoc. 30, SO3 (1931). (12) S W A L L I,. E ~C,. : I n d . Eng. Cheni. 33, 394 (1041). (13) WII.LI.ISIS.-1. \V,,.ASP \V.ATSOK, C. C . : C'oltl SpI,ii1g Ha~,bor,8J.niposi:i Q u n l l t . 13ii)l. 6, 20s (1038,. (1.4) \ V I . N . ~ X. J~ . , J R . :J . Biol. C'hrin. 90, 443 (1031). (15) \VI.AI.AS~J . . J R . :J . A n i , Clietii So?. 53, 329? (19311. ( 1 G i \ V I . M . ~ S , J . . J R . :C'lietn, Re\.,Q.19, 213 (l93G).
A PHYSICAL-CHEnIICAL STUDY O F ZEIX IS AQ'I-EOI% DETERGEST SOLUTIOSSL JOSEPH E'. FOSTER? Ocpn i , / i i i cti I o,f Pii ysica 1 Ch eiti is/i,u, Hu i,i'(/1.d dledica 1 School, B n s I o r t , .llossac/i i i s c l i s
Rccc.ic,crl .-I
iigii,sl
1 9 >194s
Ph!-sicn,l-chemical investigat ioris of zein :tnd the other prolnmines arc caompli-. cnteetl by the necessit'y of \\.oiking eit,hei;in solut,ions of high pH or in t'he pi'esence of a 1:wge proportion of organic solvmt, usuiillj. a l c o l ~ o l . ~Thid limit8at8ioriis pai,t ic\ilai*lyserious in t,he caze of electi,uphoretic measurements, n4iich can be intei,pret,ed in s~iclisolvents only v.itli consicleralile caution. This has proved n serious linntlicnp in fract'ionnt'ion st'uetlies on zein. Sonp-like sulist'ances have lieeii reported t o lie dispersing agents for zein (IG), and 4nce only lon. concentrations of the detergent w e yequired it seemed possible t,liat this inight permit electi,ophoi'esis of prolaniines in a neutral, efisentially aqueous medium. It v a s recognized from t8he~ o r of k Miller and Anderson (T), Luiitlgi,en ct al. (4, 5, G j , m c l Putnam arid Neiiixth (6, 12, 13, 14) t,hai complex forinzit ion might be espected. Study of' such coinpleses miglit prolritle much or no infoi*mntionregarding t'lic propert'ies of the zein itself, depending on the degree t o u.liic.11 the properties of the zein molecule are nlteretl or masked in the comples. For this reason :t ~~Ii~~siczil-chemicul investigat'ion of zein in detergent solut'ions \vas rindert.alien. Evidence has been obt'sined that the detergent is indeed Ptwwited at the Twenty-second National Colloid Symposiuni. jvhich was held under auspices of the Division of Colloid Cliemistry of tlie .4niei~ican Chemical Society at Cainl)ritlge, Mass:diusetts, June 23-25, 19-48. Post-doctoi,al fellow i n tlie Depnr*tment of Physical Chemistry, H a n w d RIedical School. 1043-15. Present address: Departinelit of Cheinistr,y, Iowa State College. . h i e s , 1o\vn. .kit esception t o this statenlent is the study of dielectric dispersion, as reported in the I)wceding paper of this Sytnposiuni, i n nliicli case the lo\v dieleett~ieconstant of tl:e medium is a distinct adv:int:igt,. 1
t lie
176
JOSEPH F. FOSTER
bound very tenaciously t o the zein, apparently \vitJhoutlsignificantly altering the size or shape of the zein molecule itsself. THE SOLUBILITY O F ZEIS IK DE'l'ERGliXT SO1,UTIOXS
The anionic detergents as a, clii8sswere found to he excellent8disperging agent,s for zein. A4wide variet8yof commercial preparations were investigated, only a few proving ineffective. Particular emphasis \\.as placed on three types of reasonably definite molecular st'riictiire: namely, clodecylbenzene sodium siilfonatmei (herea,fter abbreviated DBSS) , sodium dodecyl sulfnte,j and the Of t'he latter, only t'hose of higher molecwla.r weight \\-cI'ceffect ive and only ,lci-osol OT (dioctyl sodium siilfosuc,cinilt,e:)\vas st'udiecl in :my (let ail. A cn,t'ionic. detergent, Zephiran' (high-moleciilar :~ll~glrliinr~tl~yll~eiizy1:~ni nium chloride), was found to dissolve zein only very slo\vly in neutral solution :uid gumming usually resulted. However, reduction of the pH t'o bout, 2.5 gave solut,ion in a few minutes with litt81eIroiible from gumming. That the dispersing action is due t,o art)iial complex formatioti \vas indicat,ed first, by the fact t,hat' optimum solubility is limitled to :L fail-ly narrow range of det)ergent to protein ( D : P ) ratios. It \va,s found that, when tJhe D : P rat,io (expressed on a weight basis) \vas below about 0.5, escess zein remained undissolved. I n the, JI :P range 0.3-1.0 solutions exhibit'ing sparkling clarity \\-ei'c ohtninetl. When the D:P ialio \vas increased much ahove this level there \\-as observed, particula,rly in the case of Aerosol OT, a'n increase in tiirhidity. In many cases solution of zein in detergent solutions actudly protluretl a signiticant, dewease in t,he turbidity of the latter. When escess zein \viis equilibrated with detergent solutions of varying concentrat,ion a linear relat>ion\V:LS fount1 bet,\\-eent'he weight' of zein dissolved and t'he conceiitrat'ion of det,ergent, t h e slope indicating a D:P ratio of approsimately 0.5 under such conditions. This \vas concluded to tie the minimum ratio giving solubility. Such zein solut'ions were found t80t)e perfectly sta tde t,o\vartl dilut,ion \vit8tiany volume of wat.er. This behavior is quife different from t8hatof zein in the usual solvent' systems such as alcohol-n.iit8er, where solubility is limited tjo a fairly narrow solvent' (bornposition range and precipitation can be produced by dilution wit'h either solvent component. 'l'his again inclicat,es t'hat the solahility in tlet,ergenh is n result of comples formation. I.;FFP;C't'
O F VARIATIOR- OF
l" AEU'D
I O S I C STRENGTH
The solubility of zein in Duponol PC \\.as investigated over a pH range of approsimately 2 to 11, On t81iealkaline side so1ut)ionsremained perfectly clear, in acetate buffer. but on the acid side turbidity set in at a pH of approximat8elj+4.8 I n anot,hev esperiment, using DHSS as det,ergent t,he solution remained perfectly clear at a p H of' 5.6 (wetate buffer) txit c*loucled a t 1.75 (hydrochloric acid4
7
Supplicd thiwuyli t Iic (:ourt('sy of L h l'aul IJoguc of the llolisapto Clieiiiical C'oni1)any. T h e inaterial used \vas Duponol PC' supplied b y E. I. du Pont de Kcinours and Coinpxiiy. Supplied by t h r AmericRn Cyanamid and Chrmiral Cniyoration. Sripplicd by Witithi-ol) Chwnicaa1 C'oiiipntiy, Iiicl.
ZEIK IA- AQUEOUS DETERGENT SOLlJTIO1\S
177
sodium chloride). I t should be pointed out, that in all of these experiments the solut,ion was first made up in water and t,he buffer added subsequently. Zein-Duponol solutions were found t o cloud quite readily upon addition of various salts, especially a t low t'emperatore (2°C.). Solut*ion,: in DBSS were much more &able in t,his regard; hence the latter det,ergenh was used for most' of the physical mea,surements. In the ( m e of t'he anionic detergents t,hc saltingout effect was found t80be greilter t'he lo\ver the pH. The inability t,o dissolve zein in the cationic detergent, Zeptiiran, except a t lo\\. pH has heen ment,ioned. However, such solutions could be neutralized with alkali to pH 7.0 and then t,hey remained perfectly clear, even in the refrigerator. Dialysis against a neutral phosphate buffer or cixn n pH .j :icetilte buffer led t'o precipit,at'ion in t'he form of a gum. ATTEMPTS TO REGENERATE ZEIN FROM 'l"h
DETEHGP~K'I' COkIP1,P.X
&>\-era1atteniptb irere made t o recover detergent-free zein fivm the caomples, without success. Dialysis and even electrodialysis against both u~aterand 70 per cent alcohol have failed to yield products containing m o i ~than 70 per cent protein. These failures were probably due in part t o the micdlar character of the detergents themselves. Control dialyses on detergent alone showed its passage through cellophane to he very slon-. The fact that zein and the detergents have somewhat similar holubility c*haracteristics contributes to the difficulty of sepalation. Thus GO per cent acetone, which was used by Lundgren (6) foi, precipitating ova1t)iimin from the detergent complex, is an excellent solvent for zein. .Ittempts \yere mndc to find Some ion which would preferentially precipitate the detergent from solut ionh of the comples in a good zein solvent such as '70 per cent alcohol. Lead ion has proved effective in precipitating the detergent itself from such a solvent, hit in a11 experiments to date the zein has heen carried tloirn also. INTRIKSIC VISCOSITY
The viscosity of the zein-DBSS complex \\.as measured a t ii D:I' ratio of 1.0 in a phosphate buffer system at, several concenti-ations of the complex. Viscosities were referred t o that of t8hebuffer solutions. The i-esults, summarized in figure 1, indicat,ethe intrinsic viscosity of tjhe complex to be orily aboiit one-fourth a,s great, as t'hat, of zein in alcohol-wat'er. Assuming an unhydrated prolate ellipsoidal structure, this indicates a reduction in axial ratio (n./b) from 15 to 3 and a reduction in t8hefrictional coefficient' from 1.70 to 1.25, h s e d on the equat'ions of Simha (15) and Perrin (11). Almost ident'ical results were obtained in the vase of Duponol. Tn an early esperiment,, hefqre complex formation was calearly established, viscosit,ies were mea8sured on a series of solutions made by diluting 2 per cent zein in 4 per cent, detergent with the 4 per cent detergent solut,ion, so that, t81ielimiting coiiditiori gave a. D:P ratio of infinit),. The viscosities were of course referred to the diluent, i.e., 4 per cent detergent solution, and t,he concent'ration used was t'liat of zein only. These results are, given in curve C uf figure 1. The extrapolated \-a,lue nssuming linearit8yof 1,heIn vR/C U.S. In vR
178
JOSEPH F. FOSTER
plot is near that of zein in alcohol but there is evidence that the curve bends upwards at low zein concentration. There is no apparent reason why such measurements should give an intrinsic viscosity characteristic of zein, and the result is probably fortuitous. Indeed, recalculntion of these data on the basis of complex concentration, assuming all the cletergent bound up to a D : P ratio of 3, gives a ciirve almost coinciding with curve B. At first thought this reduction in a / b might be taken as indicating a split of the molecule. However, a decrease in a / b from 15 t o 5 can be accounted for in the case of a rod-like molecule just by assuming a monolayer of detergent of thickness about 20 d. on the surface of the protein. 0.3
I
I
I
I
I
I
I
I
F I G .1. Results of viscosity ~ i i e ~ s u i ~ e t i i eoii n t szein in c1odecylI)enzene sodium sulfonate (cui,ves B niitl C ) xiitl i n aqueous alcohol. For ail esplauntioti of t h e cui'vw see t e s t . BIREFRINGENCE O F FLOII'
Measurements of flow birefringence were made on zein in aqueous Duponol solution containing also 54 per cent by weight of glycerol to increase the viscosity. The studies were carried out in the instrument previously used in a study of zein in propylene glycol (2). The birefringence appeared t'o be negligible, lit'tle if any more than would be expected for the solvent alone and much less than \vas expected on the basis of previous results with zein in propylene glycol. Ho\ve\-er, measurements were complicated by a bad tendency of the solutions to foam. The results suggest the complex to be less elongated than the original zein, since the inc,reased size of the complex should increase the ease of orientability in spite of the decreased axial ratio. However, direct comparison of the magnitude of birefringence between two such different systems is not entirely safe, owing t o the fact t)hat the results depend greatly on the difference between t,he refract,ive indices of the solvent and the solute. Gnfortunately, the bire-
ZEIN I N AQUEOUS DETERGENT SOLUTIONS
179
fringence vas too low t o permit measurements of extinction angle which could be c,ompnred directly. A few measurements were made on regenerated zein, freed as complet~elyas possible from detergent (ca. GO per cent prot,ein) and dissolved in propylene g l q ~ ~ ~ l . The results n.ere very similar t o result's of measurements on t'he original zein in t,his soh-ent, so t'hnt if any split or change of shape takes place it must lie i*eatlily reversible. ULTRACENTRIFUGAL STUDIES
Numerous sedimentation st'udies were carried out 011 zein dissolved in aqueou;; detergent, solution, using an air-driven ultracenti*ifuge of t'he Pickels t'ype equipped with n modified Philpot schlieren optical spst'em. Most, of t'liese studies w i ' e niatle with a fairly well frnct ionatecl zein preparation designated zein 11-1. AIeasurements 011 t'liis zein preparation in aqueous alcohol gave a sonieivhat asymmetric peal< i\.ith S ~ O , ~of, 1.26 t o 1.43 8 in the concentrat,ion range of 2.0-0.5 per cent (extrapolated \ d u e 1.5'7). In t,able 1 are summarized some of the more pertinent results. Duponol is foiincl t'o have a sediment>at,ionconstant,, reduced t o \\-at)erat, 2OoC., of 1.0 at 0.8 per cent concentration. DBSS seclimentmecl a little more itapidly, the values i m g ing from alioiit 1.1 t o 1.4 oi-er the concentrat>ionrange O.G8 t'o 2.7 per cent', thc rate not' being a simple function of the coiicentr at'ion. The most interesting feature of the results presented in tahle 1 is the presence of only a single, usually quite symmetrical, peak atj D : P ratios of 2.35 and lo\ver. This peak has a sedimentation velocity more t>liantwice that, of detergent, or zein alone, indicating the detergent t o be entirely tiound. At D:P ratios of 4.0 and above two sedimenting component,s are present, tJlieslower sediment,ing a t a rate close t o that of detergent alone under similar conditions and presumably repre-, Renting escess unbound detergent. Furt,Iiermore, the area of the slower peak. increased with increasing escess of detergent. The extrapolat'ed value of the sedimentation constant of the zein-DB88 is uncertain but must fall in the range 3.0 or over. I n order t'o compnre this \\,it11 the value for zein it is necessaiy t o correct for tJhe differences in pnrtJial specific volume between that of zein and the comples. A part'ial specific \.oliime of 0.86 cc. per gram is calculated Iiy Ward, High, and Liindgren (17) f m n the density data of Tartar c t al. (IO) for sulfonate detergents, ivhile A'Iiller and Andet~,~on (7) report, a value of 0.87 for Duponol. Zein 11-1 in alcoliol-\vnter has n paitial specific volume of 0.78 (3). Assuming a linear relation tiet\\-eeti partial specific volume of the comples and its composit,ion, one can calculate a value of 0.82 for the 1: 1 comples. Substitution into the (1 - zip) fwt'or of the sedimentation equat'ioii gil.es a fact,or of 0.18/0.22 or 0.82, the correction in s resulting from change in density alone. The viscosity i~esiilt~s indicated a reduction in frictjional coefficient (.f,lfo) from 1 . i O t o 1.25. ilssuming no splitt'ing of the zein molecule so t,hat, the inolecular ]\-eight of the complex ~ v o ~ i l lie t l tn+e tliat, of t,he zein, one cat1 c a h i h t e a ~ x l u eof ~ 2 0 of, ~2.8 s, \rhei,eas split,ting into half-size molec.ules shCJdd lend t'o a value of only about 1.8. It should lie rioted that all the s20, \ d u e s for
180
JOSEPH F.
Fowm
the presumed zein-DBSS peak are ]vel1 u L o \ ~the 1.S value. \'dues iLt finite concentration should be, if anything, lower thax the limiting value. I t hence appears likely that no splitting of the zein molecule takes place in DBSS. The significantly lower values of s in the case of the Duponol complex are puzzling, and mould be compatible with a split into half-size molecules. I t is difficult tto imagine why of two SO closely related detergents one should cause splitting m d TABLE 1 Sedimentation results o n zein-detergent complex
___-_
____
I
DETERGENT
~
D:P
RATIO
CONDITIONS'
~
REMARKS
I
1 S?O,W
DBSS . . . . . .
1.0 1.0 1.5 2.4 4.0
Duponol . 1
I
~
__
- -
-
'
0.5 1.6 1.6
Single peak Single peak, somewhat skewed Single peak, somewhat skewed
1 0.98 1 Single peak p H 8.3, 1.04 Single peak veronal-sodium chloride 1 0.8, p~ 4.9, ace- i 1.00 Single peak 1 tate-sodium chloride 2.4 1.57 Single peak 1.3 1.81 Single peak 1 2.5 1 1.77 1 Single peak ~
I
2.91 3.38 2.84
2.4 4.0 1.6 1.6 1.6
E,
.
1
~
__-_____
~
_
_
* The
concentration is given as per cent total solute, protein plus detergent. Unless otherwise stated the buffer system was phosphate plus sodium chloride, p H 7.2-7.4, total ionic strength 0.15-0.2
the other not; f o the ~ time being it neeins preferable t o assunie the low secliinentation vadues as due t o greater interaction, solvation, o r some other factor. E L I ~ : C ' L ' l ~ O P H O R ~ : ' ~RTUDIEB I(!
hltliougli tlie priiiury pwpo;.;e of this in\-estigation was to explore the possibility of utilizing electrophoretic studies on zein in aqueous detergent as a crit.erion of homogeneit)y, it was impossible to inalce more than a few preliminary runs because of tlie pressure of more urgent) wartime projects on the equipment. DBSS was found t o give a single peak \vith a mobility of 18-19 X cmn2
volt,-' see.+ in accordance nit11 the observations of Lundgren et al. (5). At lo\\* D: P ratios most of the zein preparations gave twvo poorly resolved peaks with mobilities in the range 7-9 x and differing usually by only about 10 per (dent.. A s tlie D: 1' ratio \vas increased the inohility of both peaks increased and t'lie diffei.ence 1,etn.ecn them bec,anic less, so that at zt D : P rst>ioof 3.0 there was onlj+a slight asymmetry. In n run at a D : P ratio of 9.0, however, 3, definite split \ Y W obtained, the fast, component' having a mobilit'y of 15-1G x and pres t i n i : ~hly c.orresponding t o free detergent. Kot enough runs were made a t interAscending
Deseendhg 30
Tjmr in M i n u t e s
Standard
Fracfion
30
60
/I4
'r;
Zein
a-1
Laboratory Zein
I
L
Gliadin
w
4
FIU.2. Elect8t~ophoreticpat'tertis of various zein pi*elx~ratioiisarid one gliadin piepara-. tioii as the cuinplexcs with tlodecyllmizerie sodium sulfonate. of 1 .O i n veroiial buffer of pH 8.6 a n d 0.10 iotiic strengt 11.
Carried out, a t a D:P i~atio
mediate D: P ratios t'o determine at what ratio the detergent peak appeared. The: results were very similar to those of Lundgren et al. ( 5 ) on denatured ovalbuniiii with tlie exception of the split at Ion, D: P ratios, which was concluded t o be some indication of inhomogeneit'y in the zein preparations. This belief was encouraged by tlie failure of one zein sample, prepared in the laboratory a t low temperature, t'o show the split. This is shown in figure 2, which summarizes electroplioixetic patterns on several zein preparat'ions and one gliadin sainple,8 all cari.ied out under as nearlj* as possible identical conditions at a D:P ratio of unit'j'. While t'he difference between the zein prepa,ratioiis is not very striking in thew particular runs, it' is.true that t,he split did invariably occur in the comniei.cial zein and fract)ions prepared therefrom and never occurred in the case of the laborat'ory preparation. The gliadin preparation appears to be much less homogeneous tlian any of t'he zein preparations. SupplictL thi~uuglithe cuurtes). of Dr. 13. B.Vickci,,~.,Coniiectirut~.-lgricultaral Expeii~ n e i i Station, t S c w EIavc11, Connecticut ,
182
JOSEPH F. FOSTER
Since zein has so few potentially ionizable groups of its own, any charge tlifferences between different species must be overwhelmed by the charge contributed by the detergent. This fundamental difficult8y \vas recognized from the start, and it seems apparent that any electrophoretic resolution olitained must, be on the basis of differences in molecular weight and/or shape rat,her than charge. Unsuccessful attempts have been made to dissolve zein in uncharged anti in dipolar-ionic type detergents which would not contribute t o the mobility. This aspect of the problem is still being investigated, recent results intlicat,ing the possibility that soaps may give better resolution than the sulfonated DISCUSSIOS
On the basis of the above experimental evidence it appears that solutions of the water-insoluble protein zein in anionic detergents typified by dodecyl benzene sodium sulfonate can be pictured as containing protein-detergent aggregates. The size and shape of the dispersed unit can be approximated as unaltered zein molecules with added detergent, although a split into smaller, for example hnlfsize, units cannot be completely ruled out. The composition of the complex is fixed only within limits. A certain minimum number of detergent ions, corresponding t'o a D:P ratio of approximately 0.5, appears necessary before the aggregate is soluble in water. Thereafter the amount of bound det,ergent can be increased until a saturation value is reached. This saturation limit cannot be definitely ascertained from the present results but probably lies somewhere in the D:P range 2.0-4.0. The interesting stoichiometric binding of detergent observed by other workers in the case of native ovalbumin ( 5 ) and serum albumin (14) was not observed in these studies nor is it t o be expected. Those complexes apparently involve one detergent ion per one or two cationic groups, and since isoelectric zein contains only about twenty cationic groups per 100,000 g. (1) such complexes, if they occurred at all, would lie in the D : P range 0.07 or below, far below t,he ratio found t o give solubility. In fact, the composition at minimum solubi1it)y corresponds to approximately 140 moles of detergent per lo5 g. of protein'" and a t saturation the value must be of the order of 300-GOO. The question of how this large amount of detergent is bound cannot be answered at present. In general, the formation of protein-detergent complexes has been explained on the basis of ionic binding. Palmer (9) has, hoivever, suggested the importance of non-polar forces. It is of some interest in this connection that zein probably contains t,he highest proportion of non-polar amino acids and the lowest number of ahayged groups of any well-known protein (1). ItJ would appear that if t.he Iiinding is ionic each cationic group of t,lie protein must bind many (fifteen t o thirty) detergent ions, perhaps a whole detergent micelle. The int,eresting observation that some detergenk giving rat her turliid solutions were Unpublished nieasurenients of M r . H . Yui, Io\\.&State College, .ltuFs, Iowa. This \vould correspond to a net negative charge of 140/105 g . provided no cations w e hound. Dr. Dexter French has pointed out to t,he nuthor t,hat at pH 11, where zein 1)ecames water soluble, t h e net charge as calculatetl from the titration curve is appro\imntel>r 1050/105 g . 9
10
ZEIN I N AQUEOUS DETERGENT SOLUTIONS
183
actually rendered clear by solution of \vater-insoluble zein \vould seem to indicate a brealido\vn of t,he micelle structure with reaggregation of the detergent around the protein. Palmer suggests that the maximum detergent-binding ability can lie esplained on the basis of close packing of a detergent monolayer on the surface cf .the protein (9). It) is interest'ing t h a t the maximal binding power of zein is very similar t o that of ovalbumin. Since this research \\as c a i i e d out there has appeared a report by Lundgren and coworkers on the nature of the dissolved unit of the lieratin-detergent8 comples (17). Their complexes were quite symmetrical (axial ratio about 4 t o 1) and t,liey ol)served t'wo elect'rophoretic components (mobility about - 9.2 and -11.9 X 10-5 sec.-l volt-'). These results, in particular the latter, are strikingly similar t o those observed with zein. SUMMARY
Zein is i~eadilysoluble in aqueous solut#ionsof anionic detergents provided the rat io of detergent t o protein (by weight) is above approximately 0.5, The nature of these solutions has been investigated by the following physical-chemicd methods : viscosit,y, birefringence of flon,, electrophoresis, and sedimentation in t,he ult rncentiifuge. The physical measurements are in accord \\it11 the assumption t1i:it the zein is moleculndy dispersed and not significantly altered in shape, and th:xt t,he detergent' is 1)ountl tenaciously t o the protein. The possibilities mil limitations of t'lie electiuphoiet'ic analysis of zein preparations as the detergent, c om p I e s :ire cl isc usued. The nuthor is indebted t o Professor Etlivin J. Colin for providing the facilities of his 1:~boratoryand t o Dr. ,J. L. Oncley under whose direction the ultracentrifugnl studies i\'ei'e carried out. REFERENCE8
( I ) COHS,E . J . , . A N D EDS.ALL, J . T.: Proleim, ..Lrtiino d c i d s ntzdPeplz'dcs, Chap. 15. Reinhol(l Puhlisliiiig Cot'pwatimi, Ne\, York (1943). (2) FOSTER, J . F . , A N D E D S A L LJ., T.: J . Am. C'hein. SOC.67, 617 (1945). J. F . , A N D FRENCH, D.: J. .\in. Cliem. Soc. 67, 687 (1945). (3) FOSTER, (4) L ~ ~ N D C ~HR. El',: S , Textile Resewch J . 16, 335 (1945). (5) LVSDGRES, H . P . , EL.UI,D. \V., A N D O ' C O N N E L LR, . A , : J. Biol. Chem. 149, 183 (1043). ( G ) LITSDGREX, H . P . , A K D O'CONNELL, R. A . : Iiid. Eng. Chem. 36, 370 (1944). ( 7 ) ~ I I L L E RG, , I,., A K D .L\SDERHSON, I
