180
LCTSDGRES, STEIS, KOORN,
n - O*C'O>TELI. ~
STABILITY OF SYSTHETIC IiERdTIS FIBERS IN -1LCOHOLK,ZTER NIXTTIRES 1HEORETlC41, HASIq FOR .l S E T J I E T H O D FOR ~ O L U B I L I Z I h ' GFEATHER
r 7
KERSTIX~ HrlROLD 1'. LUXDGRES-, ASDREW A I . STEIS, VIRGISIA
M.KOORS,
AND
RICHARD -4.O'COXXELL ITesfern K s y z o t i d Research Laboratoty,Z .l/hnr,y, C'aiifoornia Receired August 85, 1947 CONTESTS
I. General consideration ui protein stability and the stabiliry of synthetic protein fibers. . . . . . . . . . . . .. 11. Force-teniperaturtx behavior of synthetic feather keratin fibers in alcohol-n-ater solutions. . . . . . . . . . . . . . . . . . . . . . .................... 183 A . Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183 B. Apparatus, . . . . . . . . . . . . . . . . . . . . . 186 C. Force-temperature behavior of feather keratin fibers in x a t e r . . . . . . . . . . . 187 D. Influence of alcohols on force-teniperaturc behavior . . . . . . . . . . . . . . . . . 189 E. Interpretation of effects of alcohols on the fiber systeiii. . . . . . . . . . . . 190 F. Influence of inorganic ions on force-tempera1 ure relations. . . . . . . . . . . . . . 192 0 . Yature of t h e non-electrostatic interactions i n feather keratinfibers.. . . . . . . 195 €I. Evidenve for disulfide cross-links infeathpr keratin fibers.. . . . . . . . . . . . . . 197 I. Conclusions relative t u force-temperature studies . . . . . . . . . . . . . . . . 198 111. Application of alcohol-water systenis t o the solubilizarion of feathers.. . . . . . . . 199 I!-. Possible application of t h e solvent systerii alcoho!- warer-salr t o the solubilization of othei proteins. . . . . . . . . . . . . . . . . . . . . . . . . . 204 I-. Sunniary . . . . . . . . . . . . . . . . . . . . 205 V I . Referenws . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206
SOLUBILIZATION O F FEATHER KERATIN
181
Hardly more than two decades ago proteins n-ere regarded as colloide of indefinite size, the properties of n-hich were determined by the state of dispersion and by the materials adsorbed on the surface produced by the dispersion. Proteins are now recognized as natural high polymers, many of which have been sho\m to possess definite molecular weights and definite compositions and to obey the phase rule in solubility; furthermore, proteins have been s h o l n to react stoichiometrically with acids and bases, with anionic and cationic detergents, and with acidic and basic dyes. Protein molecules are also now recognized a? flexible chains. These chains are stabilized in network structure as in wool, feathers, hides, and silk, or they are curled-up and stabilized in corpuscular configuration as in casein, soybean proteins, peanut proteins, zein from corn, and a host of others. But there is much yet to be learned about proteins, particularly in regard to the forces which determine their stability, including their ease of denaturation, of solubilization, of transformation into the fibrous state, and of stabilization of the fibrous configuration. For instance, interchain forces such as the disulfide cross-links in wool, as Harris (6) has shown, determine the ability of a fiber to withstand the effects of solvents such as water. The significance of molecular interaction can be stated in a general way: The sum total of physical properties of any substance is determined by the degree of mutual interaction between constituent structural units; that is, solubility or insolubility, hardness, flexibility, and tensile strength are directly related to the number, kind, and distribution of the bonds that hold the molecules together. Protein> vary widely in solubility depending on the degree of mutual interaction of their chains. Some proteins, such as .ilk. collagen, and keratin, are insoluble in the common neutral solvents. This property is desirable if the materials are t o be used directly a> articles of commerce. but it presents difficulties when u-e seek to disperse them in order to utilize them for such purposes as synthetic fibers. Even among the corpuscular proteins wr.find striking differences in solubility; some are soluble in water at neutral p H ; other%dissol1.e in neutral salt solutions, yet many others, including variow >eed and nut protein>, :ire relatively yoluble in I\ nter or neutral salt solutions, but are much more soluhlr. n hen theii aolutioni are macle alkaline. ,-Icertain few corpuscular native protein. cala>+ifiedas prolamine>, including zein and gliadin, are recognized by their soliibility in aqueous alcsohol solution>. T o be hilitable as a ran material for synthetic fibers a protein miist have an appropriate chain length. -1 protein consisting only of short chains cannot bc espectetl to give fibers of desirable quality. For instance, the protein salmine of molecular TT-eight 5000 does not form fibers, whereas egg albumin of molecular weight 45,000 can be transformed into oriented fibers having dry strengths over 2 g. per denier. The nest consideration in the handling of a protein is the fact that the solvent must not degrade the protein chain, and this is where the use of alkali has limitations. For instance, in the dispersion of feather keratin in alkali a t pH 13-14 rapid hydrolysis of the protein chain occurs, with the result that fibers prepared from such dispersions show progressively poorer quality as
tlie aging is continiid i t I - ioi the-c ic'aron- t l i a t rien :mtl milder methods for tlie dispelhion ot ~ndu-tr~all\iiitcirhtlnp m t l t l i f i c iiltly bolnble proteins .uch aicathcr keratin i i i c k i n g w i g h t C'heniical utilization oi kathcr hcratin i b onc 01 11ic' pioblems assigned t o tlle ]Yestern Regional Laboratory for investigation. Poulti y feathers are practically purc protein, arc rhcap, and arc :LT ailahle in thii c ~ ~ i i nvt in i (piantitie?eqtimated at over 100,000,000 p o ~ i n danniially. ~ 1'he attractiT? forces that htabilizc the pI(Jtel11 r h d i n ~in feathers are presumably similar t o those in other proteins 131th the ewsption that in feather-, 3': in wool, because of the large proportion of cystine, there is extensive dlhulfide cross-linking bet een neighboring chains. Hut even when these bonds are hroken by reducing agents the protein i Y insoluble m the common nonalkaline solventq. The attractive forces which remain are presumed t o he ( u ) hydrogen bonds huch as could occur between the backbone of neighboring chains or between side chains, ( b ) electrostatic bonds betneen acidic and basic residues on the side chain+, and possibly ( e ) van der Waals forces between polarizable portions of neighboring chain+. The energy required t o di-wciate the,e bonda and heparate the protein chains, whether t o rearrange them by denaturation, to dissolve them, or t o separate them mechanically, ivill clcpentl on tlie number, kincl, and distribution of the bonds; this energy nil1 J arp ~ i t hthe environment surrounding the protein. ,Just as the energy required t o -eparate the ions of a crystal of an inorganic salt in the dry condition is high compared with that In water, so is the thermal energy required t o denature a native corpuscular protein lo\vered when the protein is placed in water. For e u m p l e , in the dry condition egg albumin is stable to temperatures well above 100°C'. , whereas in n-ater it denatures rapidly at 60°C;. When ethyl alcohol is added to the Tvater the rate of denaturation is increased as a function of the amount of alrohol added. Eyring ( 5 ) has pointed out, the solvents water and alcohol act as catalysts in lowering the thermal energy required t o ctennture the protein chains Similar effrcti oi environment ~vould be expected in the -olubilization of protein.. The problem, thcii, of finding n yuitahle mean- ior -olubilizing feather kcratin ntially in finding the appropriate bo11ent ratalyst which favor3 thr dissociation of protein rhains u ithout degrading them. One approach i i through the application of synthetic detergents (10) In aqiieoiiy ioliition at neutral pH. ;\fter the disulfide bond. are reduced, the feather keratin is solubilized with anionic or cationic (letergenti through the formation of complews ot protein and detergent. The kci atin in thew complexes, as determined from measurementi in the iiltraceiitr~fuge.by difiusion, and hy osmotic prewire ha5 a molecular \\eight comparable in size t o that ot egg albumin (14) ('oncentrstrd solution. are highly \ iscolis and exhibit birctringeiice of flon * iiitlicat ing that the protein i- iinfoldcd. The holiitionh ha\ e desirable spinning propertici, upon extrusion and (wagillation the fillers which form can be extracted fret, of detergent nith 70 per vent aqueous acetone: with 5ufficient -tretch the f i h c ~ > shoiv a relatively high degree of cryhtalline orientation. The tletergent method i- applicable t o 1 arioii- protein-, thc pi~llcipal I?A\,
SOLUBILIZLiTIOX O F FEATHER ICERATIS
183
qiiiieinent being that they har e a iufficient numbei $ ) I :icidic m t l basic gioiip+ neceysary ior the p i i m ~ i yinteraction betneen the piotrin and the detergent. C'cdlapen i i an eveptioil: i t canno: i ) e 1i:anclled by the detergent method Th(.
detergent method I S iiirther limited by ~ l i cielatir ely l u g e amounts of detergent itating e\tractions \ \ h i d i a i e .lo\\ and inconi enient particularly io1 fiber- har ing laigc diametei Feathers, aitei ietliiction v i t h I)i-ulhtr or monothioglycd, can be tlissoli-et1 at pH \slue+ clo+e to neiitrality 111 a niiniher of iolrent- nhich are recognized LIS hvdi ogen-hntl (1iw)ciating agent+. Thii+, ioi e v i m p k , reduced feather5 n 111 (11~.01re in 40 pei cent iiira ~oliitions,48 per cent guanidine solutions, and 6 1 pel cent ioditim dicvlatc. .oIiition~,all of uhich :ire piotein denaturants ,Jane:, and Mecham (8) in thii Lahoratory ha\e compared the effects of detergent> nith ieveral of these agents and in general found them to t)p equally effective and t o diyperie :I maximum of about 80 per cent of the feather material Furthermoie, chemically ~ d u c e dfeathers . can also be disperbed in agents which disso1i.e >-TEMPER.%TURE BEHAVIOR OF S T S T H E T I C FE.%THER K E R . t T I S
F I l l E R S 1%- .%LCOHOL-W.\TER
M1XlTRE.S
-4.I nt Tod u et ion -.Ineffect'ix method for studying protein-solr-eiit interaction involves the measiirement of the tliermoelastic properties of fibers in the selected soli-ent. The mechanical behavior of the fibers is highly sensitii-e to variations in the solvent environment ; furthermore, the effects of temperature on the are reversible, thus niakiiig the resiilts amenable t o thermodynamic considerations. IYe haye already refei~edt o the pronouncetl influence of \vater alone in favoring tlic separation of protcin chains by the difference in stability of wet and dry egg albumin t'owarcls heat ; and similarly this stability tiifl'erence is apparent in tlic wetting of the synthetic protein fibers. For instance, uncured feather Beratin fibers which give thy stimgths of 1 g. per denier hai-e a \vet strength, in the relaxed condition, of 0.1 to 0.3 g. per denier. The first effrct of \vetting the fiber a t low temperattires is: a shortening, pro1)ably the result of attraction of water. by the polar groups it-ith the simultaneou..: fission of weak secondary interchain attractions. The ciwliiig entropy fa\.ors :I shortening of the 11-ater-plasticizednetwork chains t o a somewhat' less extentled st at e. .Ipplication of heat to the tiher in this state causes :I slight expansion provicletl
184
LXXDGRE?;,
STEIA-, KOOIW;, AND O'COXWLL
the temperature is below the critical region, which is approximately at 43°C. for a feather keratin fiber near the isoelectric point. The espansion is typical of a normal solid material. Beyond 45°C. it rather sudden change of state takes place; the fiber contracts to a relased state in which it now eshibits the thermoelastic behavior of a typical rubber-like material. lye shall discuss the nature of the forces that stabilize the network chains in this rubber-like state. .Just a i the presence of alcohol in nater loners the thermal energy necessary for denaturation of egg albumin, as \\e pointed orit earlier, alcohol-water mistures containing inorganic electrolytes lower the thermal and mechanical energy required to maintain constant length in the fibers. This effect of the solvent miltiire i* through tli:,aoci:ktion of the stabilizing uttractionh. It i h more convenient to measure changes in the equilibrium force required to maintain the fiber at constant length with changei in solvent environment than it is to measure changes in length. Furthermore, the experimental arrangtment for equilibrium force-temperature studies permits the study of stress rclasation in the fiber following rapid elongation. The measurement of stress relaxation when carried out in the same environments as the equilibrium forcctemperature measiirements also gives information regarding the nature of interactions in the fiber affected the solvent environment, The equilibrium force on a network system is a function not only of the trmperature, but also of the internal attraction* of the system malogous t o the prcssure of L: real gas. The comparison between the force-temperature of chain syztrnis xith the prrsaiire-temperatnre relation among ordinary molecules is givrn in figure 1. lYhcn iul)ber, for elample, is stretched more than 10 per cent the forre increases wit11 tcmperat ure ab indicated ; normal solid materials exhihit :I negative. temperature corfficicnt of force The slope of the force-temperature. c i i n e is :I meiiiiire of tlw r1ntropy of chain ciirling, and the intcrccpt i:, :I measiirt\ ot mterch:iin attract ion-, Figure 2 summarize:, tlic T aiioii:, foicc>>acting on a fiber net \\ark mrrounded hy a solvent Tentliiig t o clibper-c t h t nrtu ork chainb in thi9 cnae is the osmotic 1-nclling forcc in addition to the mechanical deforming force, and the thermal binct i c iorcc. Opposing the>(% forces are the attrwtive force:, holding the netnork together. In addition, tlicrt i. the amall contribution ot the hydrostatic forcc of the, solvent acting on the fihcr. 'I'hc cxorresponding rh:ingr- in cnwgy in llie tem may \)e rcprcaente(1 a- fo1lon;-: AI'>
+ fATi + ?'AS = A E + 1'Al= AH
where f A L is the mechanical work of deformation, f being the applied force and A L the resulting elongntion; AF8 is the free energy of solvation, that is, the freeenergy decrease involved in the interaction of the protein with the solvent medium; TAS is the change in kinetic energy of the system, T being the absolute temperature and A S the change inentropy; A E is the change in the fiber network energy; PAT' is the hydrostatic n-ork done, Tvhere P is the hydrostatic pressure
SOLUBILIZATION O F FE.4THER KERATIN
LIQUID
GAS S
GAS
185
SOLID
CHAIN MOLECULES €=IO%
T PV = RT P=+ = BOT
T
REAL P-S.3 =-A(V) + B(V)T FIG.1. Coniparison of states of interaction in chain systems Kith ordinary molecules. Rubber, for example, a t greater than 10 per cent elongation exhibits a positive teniperature coefficient of force ivhich charactrrizes the rubber-like state.
ATTRACTIVE
’ /
FORCES
fTt 7’ ’ //I
T t, t
HYDROSTATIC PRESSURE
and AT' the change in volume of the fiber system :i>-ociated with a changt. of state. From these considerations the relation lietween the equilihrium force and temperature for the fiber-poll ent system is deriwd as follow :
and therefore :it const ant elongation,
f=A+R?' B. Apparatus 'l'he apparatus illuitratetl in figure 3 was used to mezliiirc the. rquilibrium force-temperature and the rate of stress relaxation in the fiber. It consists of a thermostated chamber, -4, in which the fiber and thc sol1,ent arc placed. One end of the fiber ia attached t o a hook at the Iiottom ot the chamber and the other t o a stiff wire communicating the tension on the fihcr to a torsion \\.ire a b o e~ tlic chamber. The torsion !\ire, made oi tempc*red t)cryllium-coppt.r alloy, is 1-mersible t o small displacementr. The small &placements of the torsion 13 ire are magnified by an optical lever arrangement illustrated in thc diagram. .I mirror on thr. torsion \\ire reflects the i'ociiscd image of :i slit or cro-i luii. over a uniform sc~ileplaced on thc wall opposite the mirror. 13y this arrangement, for example, :I 0. I-mm. tlisplacement of I I IO-cm. htwr is magnified to LL 4-c.m tlirplacement on the >(Y&>; :ircordingly a 10-cm. f i h imtlergoing :L total di>plucement ot 0. I mni. can l)c c.on-ideretl ii- l)eli:t\ ing (> s t :in t le ngt 11. '1enipri:itures iiom 5' to SOY' :iio maintaintvl i n tlic thr.mio-t:it cliarnlwr hy ci~~c.ul:iting mixtrirc- of \\:iter from t n o bath-. otic :it 80T'. aiitl the othcr of ic'c \\ ate1 . .I nnotioii-pictui,e c.iimci.a i, u 4 t o Irlcord tlic 1 aiiiition i n i o i c r ; thia arrangcnicnt 1 - particiilnrly useful \\lien tlic measurement of the rate of st r+i rela~ntioni, desired. 'Taking into account the period ot oscillation oC t h tor-ion ~ 1 1 ~ it' . is pos>il)lr,in this manner, t o record the f o i on ~ ~the fiber at 0.1-CY. interxal\. Besides he knoivn speed ot the camerii, a hecondtlry record of the, 1 i m p j- kept by photographing the queep of L: l O - - t ~ * .\top\\atch (luring thtl c,upcrimcnt. The tle*irctl etongation on the hber is obtained by arljii-tmcnt of the height of the p1atfoi.m on which the fiber chamber rest-. This adjustment is made by shift of :L le\-er which operates, between adjustable s t o p , a cam arrangement on which thc> platform is placed.
When the decay of stress is to be followed, the desired elongation i- applied to the fiber and the motion-picture camera is started simultaneously. The initial rapid decay of stress is recorded at consecutive 0.1-see. interval exposures; later on, when force-temperature measiirements are carried out, single exposures are made from time to time as required. h t the end of the experiment the film is developed; the scale readings are measured with microcomparator; the readings are converted t o force Jvith the calibration ~ a l u e sfor the system and finally, the force or stress i i plotted as ordinate against the recorded time aq al)sci+u.
,--------
Fit:. 3 . Diagram of apparatus for mettsurenient of rquilibriurii force-trniprat uw boliavioi. slid stress relasation of single fibera. The effect of tenipcraturr and solvent o n t h e f o r w o n t h r tiher thermostated in chamber A is caommunicated b y a stift ivire t o ttir torsion \\.ire 13; thr. tlisplacrnient of the torsion wire, enlarged tiy t h e opric*allever systpiii. is rem r d d by t h r inotion-picture c m w r a . A secondary rrcord of t i n i r is kept t)?. photographing t l i r s s \ v r ~ pof a IO-sec. s t o p watch. Ilesired elongation of t h r ti1)r.r is obtaintyl by lo\vc,ririg t 1)lat fori11 n it11 a Irvrr--c)anis>.iitrin oprratiiig tictivrrn a d j u s t a ~ ~stops. lr~ hr1
0. I"orce-tpnipct.atcrr.1 bchnaior of
fmthpt.
keralin ,Iihos it1 wafer
Figriw 4 illustrate* L: typical relasation curve for the keratin fibers i n
atcr in the iwelectric region at approximately pH 5 . The upper curve is R rccorci of the ehange in tension on the hber n-ith time, showing the initial rapid fall and the - u k c l u e n t approach to a state \\.here the force changes so slon.1y that it can be con4dered essentially constant. In this constant rrgion the force at ocpilibrium on the fiber network system a t constant elongation is a meahwe of cnrrgy required to displace the net\\-ark chains in the solvent and at the temperalure stutlictl. The variation of the force with temperature is reversible. ('haructeriitic of a rubber-like material, the force decreases when the temperature is tlecreazed. Because the system is reversible with temperature, thermodynamic i\
188
LLTDGRE?;,
' 0
0
STEIN, ROORN, .4ND O'COh3-ELL
IO0
200
400 MINUTES
300
FIG.4. Relaxation of stress i n R sq-nthctic fcatlier keratin fiber follotving rapid elongation, and the effect of tenipernturc, on t h r equilibriuni force a t constant length. Following the initial rapid relasation t h e stress changes slowly ivith time and can be considered essentially a s constant. T h e poFitivc- temperature coefficient oi force indicates the rubherIilce s t a t e of the relaxed fiber.
SOLUBILIZATIOX OF FEATHER KERATIN
189
lased feather keratin fiber in water in the isoelectric region over the temperature range 5°C. to 80°C. The average diameter of the dry fibers used for this investigation was 0.013 cm.; the average wet diameter (relaxed) was 0.024 cm. The course of relasation of the fiber to this rubber-like state can be followed in the same apparatus in another manner. The wet unrelased fiber is attached in the chamber in the solvent belon- the critical temperature region a t , for esample, room temperature, and the force is adjusted to zero or to a low value. Figure 5 also illustrates the transition in state of the fiber on relaxation. As the temperature is raised, the first effect is a slight elongation of the fiber, typical of thermoelastic behavior of "normal" solid materials, but then as the temperatiire approaches the critical region around 45"C.,the force on the fiber suddenly increaser as the constrained fiber tends to contract as shown in the figure. The cyuilibrium value of the force is reached as the fiber becomes rubber-like. It i5 apparent from the figure that the fiber shows a marked hysteresis on cooling; only a slight deviation from the straight-line hehavior is apparent below the transition temperature, xhich is interpreted as clue to recombination of the alined chains. Presumably on long standing in the cold, further chain interaction would take place to give the state similar to that of the original unrelaxed filler. For the experiments which follow we have adopted as a standard state the fiber relased in boiling water. We shall compare the effects on the system of added alcohol,+,inorganic electrolyte, and finally reducing agent.
I). Injluencc of alcohols on forcr-tenipcl.afurr behavior 'I'he cquilibrium force is markedly affected by the addition of alcohols and clwtrolyteh to the system. Figure 6 illustrates the influence of added ethyl alcohol on the fiber-water system. Bs previously, the fibers are in the isoelectric region ; the elongation is 7 5 per cent oi-er the boil-relaxed length.3 -At constant elongation of 7 5 per cent the influence of the alcohol on the equilibriiim force is fourfold; first, at low temperature&and in all concentrations of ndtletl alcohol, the equilibrium force is high I' than the corresponding values in I\ a t w alone. The hignificant teature iq the rieyaizve temperature coefficient of iorcc. Second, at temper:itures aboce an apparent critical temperature region, vhicli is around 40" to 5O"C., and in concentration5 of alcohol below 80-90 per cent concentration by weight, the equilibrium force iq Zower than the correhponding ydiieb in xater. Furthermore, the values &crease progressirely as the coilcentration of the alcohol z~zrreascsto about 50 per cent. The principal effect oi the several alcohol concentrations under these conditions is on the intercept i-aluei of lhr force-temperature curves; the .;lopes are practically the same and similar to the I d u e for water alone. Third, at alcohol concentrations abow 50 per ccnt the equilibrium force increases and becomes greater than the value in water :IS the concentration of alcohol approaches 100 per cent. The fourth Charigr in the elongation of the fibers Bill change the slopes of the curves, but t h e relative difiercnce for different alcohol mixtures is not significantly altered. For all of the studics rt,ported in this discussion the fibers were comparcd a t similar elongation
08
W t % EtOH
GRAMS FORCE 07
06
0.5
04
03
0.2
01
10
20
30
40
TEMPERATURE
50
60
"C.
70
80
191
~OI.UBlLIZ.1TIOX O F FE.1THER KERATLV
E . I/it( prc talzori of Ihe eflccts o j alcohols on keratzti ./ibcrs I n the light oi thei~motlynamiccondder:i1ions the negative temperature cocfficient of force exhibited liy the fiber s p t c m below the critical temperatiirc. i n iolutions of the three alcohol< indicates normal solid thermoelastic behavior. It apparent that the net work chains have undergone extensive aggregation. The higher force required to maintain constant length in the fiber in the new stalc indicates that the new condition favors curling of the chains. The contribution to the force is from both a change in the internal energy and a change in the en14
50% ETHANOL
'
50% n- PROPANOL --
I 2oo0
10
20
TEMPE~RATURE
30
50
€0
I
70
80
oc
FIG.7 . Influence of aqurous methyl, ethyl, and n-propyl al(-oliolson the force-temperature behavior of a rclased synthetic feather keratin fiber. A critical region is apparent in t h e alcohol-water niistures n.hich shifts progressively with change of solvent from methyl t o ethyl t>opropyl alcohol. Rclo\v t h e critical region the fiber cshibits a n e g a t i v e temperaturc coefficient of force. The slopes as \vel1 as t h e intercepts of the (aurvcs vary progressively w i t h change of solvent. .\hive the rritical teniperature region t h e alcohols progrrasively lower the equilibriuiri forrc. thc principal effrct being 011 the interrepi of t h e force-f empcraturr: ('urve.
tropy of the system. The interactions statloilizing the aggregated chains decrease progressively in changing from methyl to ethyl to n-propyl alcohol. The nen- state of interaction in the fiber is comparable to gel formation in solution. .is we shall see later in this discussion, gel formation does occur in solutions of feather keratin in alcohol-\\-ater mistiires helo~va similar c,ritical t emperat'ure region. Further similarity in the behavior of' the fibers and the solutions of feat,her keratin is evident from the influence of added urea. In hoth cases added iirea reduces the extent of interactions until the fibers remain rubber-like over the entire temperature range within Tvhich they can be studied :mtl the keratin solutions similarly remain liquid.
192
LVNDGREK, STEIK, KOORN, AND O’CO~SELL
Above the critical temperature region the fibers have rubber-like properties in mater as well as in solutions of all three alcohols below 80 per cent concentration by rveight. The main effect of the alcohols is on the intercept values of the force-temperature ciirre, This indicates that change in the internal energy of the fiber system is the principal effect of the solvents at these temperatures. This change is accounted for by interaction of the alcohols with the protein, with resulting dissociation of the attractions which stabilize the fiber network. Hecause progressively lower temperatures are required to maintain constant length in the fibers when thc solvent is changed from methyl to ethyl to npropyl alcohol, we conclude that these alcohols, in this order, are progressively more eflective in dissociation of internetwork attractions.
4-
10
20
50
Y)
TEMPE~ATURE
*c
60
70
-
c
80
FIG.S. Influenre of added salt or1 t h e force-temperaturr brhavior of relaxed synthetic. feather keratin fibets in water and in 30 per rent aqueous ethyl alcohol. Added salt lowers the equilibrium forcr i n watrr and the aqurous alcohol niivtures m e r the range of trmperat u r e indicated.
1’. Ir~flur%ct o j znorgarric
aoiis
011
jorcc-tempt rufure relatioris
The equilibrium force on thc hber both in w:itc.r m t l in the several alcohols i- markedly affected by atltlcd electrolytes. In the preqence of salt in low concentration the equilibrium force, in either case, is lowered. For example, figure 8 illustrate3 the influence of lithium chloride and potassium chloride of ionic strength = 0.1 on the equilibrium force in Tyater and in 30 per cent concentration by weight oi ethyl alcohol. In both water and aqueous alcohol decrease in force occurs above, a5 well as belon-, the critical temperature region. The effects of the alcohols and electrolytes are reversible; upon their removal by washing with water, the force-temperature behavior reverts to the characteristic behavior in ivater. The effect of salt in altering the equilibrium force in the isoelectric fiber suggests that \warv draling here with the stability of interchain salt linkages,
S0I;C‘BILIZATION OF FESTHER KERSTIS
193
subject to similar variation in activity coefficient as are soluble dipolar ions, amino acids, and native proteins in the presence of electrolyte ions (4). The interaction between ions and dipolar ions is known from both theory and experiment to vary with the ionic strength and dielectric constant of the solution. This behavior is accounted for b y the theory of Scatchard and Kirkwood (12) and Kirkwood (9). According to the theory the change in free energy due to electrostatic interaction between dipolar ions and inorganic ions should vary directly n-ith the ionic strength and inversely with the square of the dielectric constant. For example, the folloTying expression was derived for the free energy of interaction of glycine with inorganic ions:
in which y is the activity coefficient of the dipolar ion, S its solubility in salt solution, and SOits solubility in pure water. D is the dielectric constant of the solution and T is the absolute temperature. R is the average dipole distance, and a is the “collision radius,” that is, the sum of the radii of the dipoIar ion and the inorganic ions. 2 is the ionic strength in moles per liter. When the free-energy change is measured from the solubility ratio at higher ionic strengths a second effect enters in, which must be taken into account, so that the solubility behavior represents only those changes due to electrostatic forces. This secondary effect i4 the salting out of the dipolar ion b y the inorganic ions. Accordingly, a salting-out term must be added to the logarithm of the solubility ratio. S o w t o come hack again to thc behavior of the fibers as a function of ionic strength and dielectric constant, where if the foregoing considerations apply, the equilibrium force, which i g decreased in the solvent mixture, should vary similarly irith the ionic strength and with the inverse square of the dielectric constant, but* t\.ith a negative slope. This behavior i.. observed under limiting conditions as shonn in figur~9. This figure is a three-dimensional plot of the variation of the equilibrium force ratio with the ionic strength on one axis and the dielectric constant ratio on the other. D Ois the dielectric constsrit of p1re n-ater, and D i; the dielectric constant of the solvent under conderation. The equilibrium iorcc ratio, f SO,i c the ratio of the force, j , measured in the solvent under consideration to SO,the force in n-ater +elected a5 the qtantlartl condition. Starting at the upper left of the figure we see that at low ionic strength the force decreabei linearly ah a limiting condition with the inverse q u a r e of the dielectric con-tant of the d v e n t . Significantly, the force T ni*ie; independently of the alcohol type. Similarly Tritli increase in the ionic .trength the equilibriuni kor cc‘ fall>; the deviation from linearity is probably cluc to r l r salting-out eflect oi the ions on thc protein. K h e n the x-arintion of tllc iorcc vith alcohol is compared at ionic -trcngih 0.1, it i b ieen that although the limiting behavior appear- to he linear, the behavior tliffcr.> from that in low ionic 5trengths by an apparent -pecific cffcct for each :~lcoliol. It is concliiLlccl from these consideration; that electrohtntic interaction. are involved irl both lo\\ and higher qalt conccntraticin interaction involve\ premmably ttie salt linkage connecting neighboring (,hain,. The severance of salt linkages hy the intrxivtion i v i t h inorganic ions can hc cwiiidc~redas follow : /’
SOLUBILIZATION O F FEATHER K E R h T I N
195
C. Nature of f l i e norz-electrostatic interactions in feather keratin fibers (1) van der Waals attractions
Feather keratin contains a relatively high proportion of leucine compared with many proteins; it is possible that non-electro3tatic attractions of the van der Waals type between the hydrocarbon residues are affected by the alcohols. As we shall see, however, the influence of the alcohols is not specific for feather keratin alone ; other proteins with smaller proportion of hydrocarbon residues are similarly affected by the alcohols. On this consideration i t appears doubtful that the principal effect of the alcohols in the fibers involves the van der Waals at tractions.
(2) Hydrogen bonds In view of the similarity in the effect of the alcohol-water mixture on the fibers and the influence of urea, it is inferred that hydrogen bonds in the fiber are involved, urea being recognized as an agent which attacks hydrogen bonds. In considering hydrogen bonds as principally affected by the alcohol-water system, we are confronted with differences in the effect of methyl, ethyl, and propyl alcohols. The energy of hydrogen bonding involving the various alcohols would not be expected to differ significantly; furthermore, on purely geometrical considerations the small differences in size of methyl, ethyl, and propyl alcohols would not seem to account for the specific effects observed. It is possible, however, to account satisfactorily for the specific effects of the alcohols on the basis of the influence of salts on the activity of alcohols in water solutions. It is known that added electrolytes increase the activity of alcohols in water as a result of selective interaction between the ions and the solvent (2, 3, 13). Because the water molecules are more polarizable than alcohol molecules, the water tends to move into these regions where the electric field is greatest. This tendency of the water molecules to cluster around ions results in a displacement, i.e., a salting out of the alcohol with the result that the activity of the alcohol in the solution is increased. That the increase in the activity of alcohol favors its interaction with the protein is evident from the following experiment. When washed feather keratin fibers or feathers are dried to constant weight at 105°C. and then placed in absolute methyl, ethyl, or n-propyl alcohol a non-specific interaction between the protein and alcohol takes place. The combination is stable to heating a t 105°C.; the keratin binds in each case approximately 0.5 per cent by weight of each alcohol by relatively strong attraction. If the protein-alcohol material is placed in 15-ater,the m t e r displaces the alcohol so that when the keratin is again dried a t 105°C. the weight comes back again to the original value. Other proteins combine with absolute alcohol, including silk and wool as well as the carbohydrates, cotton, viscose, and pectin (7, 11). The interaction of the alcohols with the carbohydrates, in particiilar, give3 further evidence that alcohols interact through hydrogen bonding. The apparent specific effects of the force at equilibrium on the fiber system
of methyl, ethyl, ant1 n-propyl alcohol? in vater solution i b :Lccounted for on the ha& of the necchsarily increasing tendency for thew nlcohols to be salted out, in this order, from wllition. It is n-ell knonn that iiicreahe in the hize of a hydrocnrbon residue of a \libstance causeh i\ pronounwd increase in the tendency ot the material t o be salted out from solution. A2ccordinglywe interpret the influence of tlic’ alcohols in water mixtures a-, dependent on their activity. The non-electrostatic interaction Jvhich takes place probably involves hydrogen bonds. -4s a rewlt of the interaction the equilih-
25’C.
I
0.6 -
0.5
-
0.4
a
SHAW
EtOH
BUTLER
- Wt.%
FIG 10. Variatioti i n activity of ethyl alcohol n i t h increasing alcohol concentration. Addition of salt t o ethyl alcttrhol solution raises thc a(itivity of the alcohol, owing to t h e salting-out effecst Thc data a i r from Shav and Hutlcr
rium force necessaiy t o maintain constant length in thc fiber i- lonereti. The reaction can be nritten a i follon-: I
XH
c= 0 -I _ _ _ _
-
2R01I +,/
4SH
013 0 I l O l t
/
It
cI/ ,
Similar reaction of alruliol~\\ itli hydrogen-bonded m i n e residues can ocviir also The addition of balt to the alcohol-water niixturra favors this reaction through its tendency t o d t out the alcohol. T h e influence of lithium chloride on the actkity of ethyl alcohol in IT LLtcr is illiibtrated in figure 10. The data are from S h a x and Butler ( I 3). We conclude therctorc that fcntlier lccratin combincs n-ith alcohols, presumalily through hydrogen bonding, and that this interaction in water-alcohol misture3 is favored by salt, ivliicli increases: the activity of tlic alcohol through the snlt-
SOLCBILIZATION O F FEATHER KICRATIN
197
ing-out effect on the alcohol. The increase in eft'ectiwness of methyl, ethyl, and n-propyl alcohols in this order in the interaction with feather keratin i.; attributed t o their increasing tendency t o be salted out from solution. We further conclude that the interaction of alcohol< in the fiber severs interchain attractions, with the result that lower force is required t o maintain constant length in the fiber. Experiments indicate that feather keratin also react:: with the salt. The resulting severance of thr iwlt linkage:: iimilarly lower.; the equilibrium forcc on the fiber.
H . Ecideticc for disitljidc crosa-lids itz feathcr ?;crafzujibcrs So far in this discussion i r e have considered the effect5 of alcohols and clectrolytes on the fiber system. It is apparent that there are attractions still
FIG.11. Effect of added reducing agent on the keratin fiber-solvent system. ILtwnperatures ahove the critical region the added reducing a g e n t , in this vase 0.1 per cent monothioglpwl, rapidly lowers t h e f o w c on thr. fiber anti t h e fihcr tiissolvrs i r i t h e alcohol \ratersalt m i s t u r r .
present in the fiber; otherwise it ~ o u l ddissolve in tthe solvent mixture a h v c the critical temperature region. The 50 per cent propyl alcohol solution containing 0.1 lithium chloride does not dissolve the fiber even on heating tlie solution t o the boiling point. Seither does the fiber dissolve when urea is adtled. On the other hand, when a, reducing agent such as monothioglycol or bisnlfite is added, the fiber dissolves rapidly in the alcohol-water-salt mixture at temperatures a h o w the critical region. Figure 11 illustrates the effect of the added reducing agent, in this case 0.1 J/ moiiothioglycol to an ethyl alcoholirater-salt mixture. This agent was added below t,he crit'ical region and the temperature was rai.ses1. -4s seen, the force falls rapidly to zero and the fiber dissolves soon afterwards. On cooling of the solut'ion, the protein precipitates in amorphous form.
198
I~CXDGRES, STEIS, KOORS,
ASD
O’COSSCLI,
It is apparent from the behavior of the fiber that reducible bonds are present which assist in maintaining fiber structure. Considering the high proportion of cystine in the keratin, it is inferred that these bonds are disulfide cross-links. This belief is confirmed by the folloiring analysis: Before reduction there is no evidence for free sulfhydryl groups in the fihers according to the nitropruqside reaction, nhereas following trratment with cyanide a strongly positive reaction for free sulfhydryl groups in the fiherh is obtained. We conclude, therefore, that disulfide cross-links are present in the fiber which help to maintain the filler network structure .5 I . Conclusioiis rrlataw to forcc-tmpcrafiire studies In the foregoing discussion of the force-temperature behavior of uncured synthetic feather keratin fibers in the isoelectric region, we have shown that at least three types of interchain attractions stabilize the fiber network structure.
HYDROSTATIC PRESSURE V
V
THERMAL FORCE
OSMOTIC
FORCE
These interactions as illustrated in figure 12 include ( 1 ) electrostatic interactions, presumably between acidic and basic groups on the protein side chains, (a)non-electrost atic interactions, apparently hydrogen honds Ivhich probably involve t,he bnck’uoiie as w l l as the side-chain interactions, and ( 3 ) clisulficle cross-links between cysteine residues. Complete separation of the keratin fiber net,n-ork is possible \vhen a reducing agent is present. Figure 13 gives a cjualitati\-e description of the influence of the solvent system alcohol-water-salt-reducing agent, on the xnechnnicul work, fLL, required t o 5 In this connection it is significant that altliough apparently all of t h e sulfhydryl guoupa are oxidized in t hc fil)trs;. t Iir contributions of disulfide caress-linkages t o the fiber wet strength is six:tlI. It appears that only a relatively sniall proportion of t h e disulfide honds preseiit a r c cffrct ive i n cross-linking the n e t x o r k ?hains; the reniaindrr prohal)ly vonnect seyrwnts along the simic chain and arc not rflpvtivr in etahilizing thc nr:tn.ork structure. Further investigation of these renctions ma? I(anti to means of increasing the proportion of effective disulfide cross-linlib.
SOL~~BILIZATIOX O F FE.\THER
I, h i t only I\ hen sufficient reducing agent is present. The appurent rapid oxidation of d i e sulfhydryl groups in thr protein is characteiistic for thia protein. I n thr rctliicwl form the solubilitj, of the keratin reaches a masimnm in the alcohol\i:\tcr mixtures in the neighhborhood of 50 per cent concentration by weight ot alcohol, and also increases. within a limit, with increase in salt concentration. ,It higher salt concentration the characteristic salting out of the protein from solution takes place. The maximum value of the bolubility i\ difficult t o determine bec:iuse of the tendency f o r gelation in higher concentration of dissolved protein. Thc gel temperature approaches the boiling point of the solvent around 12 per rent total dia.olved protein. The recovered protein is of high molecular weight, as determined b y osmotic prewirc anti diffusion; the average is at least as high as measured for the keratin cliyperkrd by the detergent method (14). Discussion of the physical-chemical rhsuucteristics of the alcohol-water dispersed keratin J\ ill alm be presented in :L 1:iter discussion. Further insight into the mechanism of the interaction of alcohols with feather keratin is provided from comparison of the rates of dubilization of the recovered protein in methyl, ethyl, and propyl alcohols. For these experiments 0.2-g. sample- of the protein were added to 10-cc. quantities of the solvent. The alcohol concentrations \\ere 50 per cent by iveight and each contained 0.1 S lithium c-hloricle and 0.2 .V monothioglycol. The protein d i s d v e s most rapidly in the propyl alcohol mixture. On comparing the temperature at which the protein dissolves with equal rates in the three solutions we find that for propyl :ilcohol :it 45°C. the iatc i imilar t o that in ethyl alcohol at 60°C. and in methyl :tlcohol :it TO”C. Xcrordingly, the free energy of activation for solubilization clecreaw5 progressively in the methyl, ethyl, and propyl alcohol mixtiur- in the 1at io A ~ ~ t x F : - L F ’ ~ ~ :prA f = i ’ n1:0.97:0.93
rIh(l i ; i ~ e -of stre relaxation of the keratin fibers arc similarly influenced by thebe sollent mistures. The rate measurements, in accord with the equilibrium iorce-temperature and solubility measurements, confirm the viexy that the alcohol-n-ater-salt-solvent system is a catalyst for the solubilization oi keratin in thr kame sense that alcohol-water mixture,, ab mentioned earlier. are c:italy-ts tor the denaturation of native corpuscular proteins. So far \\e have diwissed the comparative effects of methyl, ethyl, and itpropyl :dcohols on the keratin. Certain other alcohols are apparently more etfecti\ e in their interaction \\ ith feather keratin. -1particularly interesting alcohol, in thiq regard, i y l .3-glycerol dichlorohydrin, compared, for example, itli iwpropyl alcohol :
CH2 C1 II c: OH CH2 Cl
C‘H3 I
H C 013 CH,
202
LTSDGRES,
STEIX, KOORS,
ASD
O’COSSELL
Glycerol dichlorohydrin combines firmly u-ith the keratin, as is apparent from the experiments which f o l l o ~ . Isopropyl alcohol, on the other hand, is less firmly bound ; it behaves similarly to n-propyl alcohol nith feather keratin. Glycerol dichlorohydrin is not completely miscible with 11-ater;it forms tn-o phases, the water phase containing about 11 per cent of the alcohol. K h e n the alcohol is added to a solution of feather keratin at p H i , the protein is extracted into the alcohol layer. This transfer is favored by the addition of salt in accord Tvith the effect of salt on the activity of alcohols discussed earlier. Other proteins,
FIG.15. Logarithmic plot of the partition of bovine serum albumin h e t n e c n mater and glycerol dichlorohydrin The higher concentration of seruni albun in in the alcohol phase (saturated with water) than in the water (saturated mith alcohol) is attributed t o interaction in solution between the alcohol and the protein. The partition is of the form y = csn,forwhichc,thepartitionconstant,is0.55andn = 1.3.
for example, serum albumin, are similarly extracted from the aqueous phaqe by this alcohol. The partition of serum albumin by the water-glycerol dichlorohydrin system is illustrated in figure 15. That glycerol dichlorohydrin i t rather firmly bound t o the proteins is shown from the fact that complexes are precipitated on the addition of sufficient salt to the solution. These complexes, qimilarly to the protein-detergent complexes, are highly plasticized and can be pulled into fibers. Alqo similar to the proteindetergent behavior is the apparent limiting stoichiometric interaction. R i i t
SOI,URII,IZ.\TIOS
203
O F FEATHER K E R A T I S
\\-here the ratio of detergent to protein varies in proportion to the number of acidic or basic groups, the proportion o f t h e alcohol bound is several times these values. Thus, for feather keratin the ratio approximates one molecule of the alcohol for each five amino acid rebidueq. n-hereas for serum albumin the proportion is one alcohol for every three residue.. The range over which this limiting combination takes place is evident from figure 16. in nhich the percentage nitrogen in the dried precipitated complex is plotted against the amount of glycerol dichlorohydrin added to the solution. The behavior of egg albumin is albo shown for comparison. The mole ratio in thih case i. 1:2. The molecular n-eight of the keratin was considered as 40.000, equivalent t o the value estimated earlier in studies on the protein-detergent complese.: (14). The pure proteins, feather keratin and serum albumin, have similar nitrogen content, namely 16.1 per cent. 14
%N
1
k
I3
I
1-L-
/
_ _ ~1‘
~P
FEATHER KERATIN a
-I
-+-I
I
4
I
0
--__rt
l
(16 I % N) 0--1_---x---+
1
I
I
Frc. 16. Apparent stoichiometric interaction of feather keratin, serum albumin, and egg alhuniin w i t h glycerol dichlorohydrin. Titrogen content of precipitated complexes from solutions of the three proteins treated with increments of glycerol dichlorohyclrin. T h e hound alcohol is in apparent stoichiometric proportion.
The value for egg albumin is 15.6 per cent nitrogen. The average amino acid residue weight for all proteins \\-a5 taken as 115. The dichlorohydrin used nas freqhly distilled and the protein solutions n-ere pH 7 . The complexeq were precipitated by the addition of exceqs lithium chloride, or alqo by qhift in pH ton-ard the isoelectric region. Examination of qolutions of mixture. of serum albumin and glycerol dichlorohydrin by electrophoretic analyses, honever, did not disclose combination between the protein and alcohol. I t appears, then, that the interaction occurs under precipitation conditions. That combination of glycerol dichlorohydrin TI ith the protein. involve5 Qecondary interaction is inferred from the fact that no chloride is liberated and the pH of the solution3 does not change. Furthermore, the complexe. decompose on continued washing with freqh water or u-ith ethyl alcohol. The so1wnt.i
204
LL-SDGREX, 8TEIT, K O O R S , .\SD O'COSSCLI.
nppnrcntly replace r l w dichlorohydrin through nias*-nction effect. C;lyc.erol dichlorohydrin pro1uti)ly interact< TI\-ith the protciii through hydrogen bonds. Significantly, glyceiol dichlorohydiin T\ :tb foiintl to di-;.oli.c nylon; the interaction in thi> rase can only in\ u l w hydrogen 1)onds. The greater attraction oi the tlichloroliythin lor the. protein- is pro1,ably relattxl to the presence of tho e1ecti.on-attracting clilorinc :itom, TTliich tend to render the hydrogen of the alcohol hydroxyl niurr clectropo-itive. Further research along these lines may kat1 t o the del-elopmcnt of desired \rater-insoluble plaqticizers for proteins. Tlic difficulty it-ith the clichloroh\-di~inin this connection is its twdency t o decompow with time, giving free hydrochloric acid. IV. POSSIBLE; IPI'I,IC.ITIOS
O F THE bO1,VEXT 61
TO THE SOI,U3II.IZ.ITIOS
\I A\T,LOHOIA-TV\TCR-S.\Ll'
OF OTHCIZ P R O T C I S S
1T-e have already indicated that the solvent syhtrin alcohol-^\ ater-salt is not necessarily specific foi, rc>duced ienthei keratin. -1s a matter of fact, mixtures of alcohol and water are recognized solvent6 for the iollon ing prolamines: hordrin irom barley, secaliii irom rye, zein from corn, and gliadin from wheat. Thece proteins readily clihsol\ e in alcohol-wter mixturei and are apparently not denatiired until heat applied. Other proteins, the lilood proteins for example, are solul)le t o a limited estent in their native form in alcohol-water mixtures provided the temperatiire is in the neighborhood of 0°C'. On the other hand, the alcohol-mter-salt solvent mixture at elevated temperatures will dissolr-e other proteins t o a gre:lter or l e ~ extent.' ~ ~ r With ceT.era1 protein.; examined in thi. isoelectric region, the tlenatiiretl protein coagu1ate.s or form, gels ~ r h mthe solution. arc cooled belo\v the respective critical temperature regions ur TI heii the amount of dissolx et1 protein ic greater than 10 to 12 per cent (see figure 17 1 . Thc-e grh. k d a r to thc fCat1ier keratin gel., .ire tlicrn~orerei~ihle.The di-solred cicnntured protein in cach ca>c uppe:irs to he 01 high molrculsu n-cight and nccordnigly 4~oultllw siiitalile for -ynthctic tihcr.. The three protein., feathcr heratin, iqinghss cdlagen, nncl pcantit protein, compared in thc figurrt were measured in 50 pcr cent ethyl :~lcoholsolution5 in their isoelectric region and at O O Y ' . Tlic .oliil)ility i? plotted a q a function of the ionic strength of the eolut ion -111 t h r w of the protc>iii- t l i b i o l ~e in the solwilt mixture. The d i i h i l i t y in cach ca-e increace4 abruptly irith increa-e in ionic ytrength ot lithium chloritlc, oning to the salting in of thr piotcin In thc intermediate zone of salt concentrution d i o ~ r nthe , amoruit oi di--ol\-cd protein i. greater than 1I t o 12 per cc>nt :md thc d u t i o n - arc grllrcl a t thi- tcmperaturc and pH . i i d with the alcohol nuxtuic. -It higher wit concmtratioii the mount of tli-4ved protein dccre3.c- t o l t w th:w 11 t o 12 per cent, on-ing t o the -alling-oiit eti'ect on the protch 'l'h(,po--ii)ilitiv oi gcncr:lil application of the -011 cnt .tcm a1 4 t tor tlw recovery ot difficultly ioluble protein. for inch;trial u
.>
7 J l i g e n w n b (.J c'olloici >(XI 1 , 539 (1'3 alcohol, n a t e r , and q d t o nix potato glob
h H b fJq:i'T\ C d r l J l t ' I J f l Z l l i g
d t e d of r l l l ~ f u ~ Y 'Of S
. c'asein, ttiiLl h ~ ~ n ~ o g l o l l i r i
SOLI-BILIZ
inox
OF FE LTHER I~ER.YTIS
205
investigated. It ih espected that the method nil1 be more effective in certain cases than in other.; numeroui factors are involved that determine the ,solubility of the proteins, among which are those indicated in thih discussion : namely, the activity of the alcohol, a i influenced by salt, the relative magnitudes of the salting-in and salting-out effect. on the protein, arid the activity coefficient of the salt. Sererthelesh, it i, forewen that the solvent 5yitem will be applicable to certain protein tern5 w c h a s feather keratin and perliaph to certain collagens for which other mclthods arc' less practicable. These experiments, together u-ith e\periment. on the filwr-qinning propcrties of alcohol-soluhilizetl protein\, alw in prqre-, TI ill 1x1 presented i n a later discussion.
W%
EtOH BY WT. AT 60°C.
1;1(;. 17. Maximuin solubility of reduced feather kcratin, isinglass collagen, an11peanut protein in50 per cent by \reight ethyl alcohol--u.ater nit11 increasing salt conre~itratioii. -111 three proteins dissolve i n the solvent niist ure. The solubility in each rase incrcasrs abruptly wit11 increase in ionic strength, owing t o the salting-in of the protein. In the intcrinediate zone of salt roncentration shown, the aniouiit of dissolved protein is greatrr t,hari 11 to 12 per cent, a i d thc solutions are gelled at this tcniprraturr and pH and with this alcohol inisturc.. ;it higher salt concentrations, the amount of dissolved protrin clcc~rc~a.ws t o less t h a n 11 t o 13 per crnt as a result of the salting-out cffrct on thc protein. bU\I\L411Y
General consideration ha- been given to thc' stwldity of protein? and 3ynthetic fiber.; the limitation. of w ~ e r a lmethod. for .olnhilizing protein-: J W ~ O nient ioned. -1method for the mea~urenieiitoi the eqnilihriuni force-temperature heliar ior of single fibers has been described. The method Tvab applied to the stiuly of synthetic feather kcratin fiber- in alcohol-water-salt mixtures. Evidence \\-a*given for. the f olloning three type5 of interactions which y t a bilize the hber netnork structure : ( I ) salt linkages, which interact with inorganic ions in conformity n-ith the theory of dipolar ion-ion interaction; ( 2 ) non-electrostatic interactions, presumably hydrogen bonds, which interact with the alcoholtj, methyl, ethyl, and rL-propyl (favored b y incr e in activity of the d-
206
LTSDGRCS,
STEIIS, KOORS,
ASD O'COSSELL
coho1 by the addition of salt) ; and (3)diwlfide cross-links, n-hich interact rvith reducing agents. The increahing specific interaction obsen ed ith methyl, ethyl, and ?a-propyl alcohols is accounted for on the basis of their increasing tendency to be salted out from solution. K h e n reducing agent is added to the keratin fiber in the alcohol-water-salt misture, the equilibrium force falls to zero above the critical temperature for the mixture and the fiber rapidly dissolves. -4pplicntion of this solvent system to the solubilization of feathers at neutral pH has been given. Similarly to the behavior of the fibera, the solubilized protein exhibits a critical temperature region below n hich the solution hets to thermoreversible gel. The recovered protein is of high molecular \\eight and a> such is suitable as n raw material for synthetic fiber,. Illustration of the solubilization of isinglazb collagen and peanut protein by alcohol-ivater-salt mistures has been given. Discussion has been given of' the iormation, isolation, and composition of complexes between feather keratin, ierum albumin, egg albumin, and glycerol 1,3-dichlorohydrin. VI. REFEIZES (1) ~ R L ~ GF. : J. . Am. C'heiii. doc.. 52, 2 33 (19301; 54, 4130 (10321. ( 2 ) RCTLER,J. .1.\-., .%sl) T H o \ r s o X , 11. \\ : P r o ( , . Roy. SOC. (London, A141, 86 (10331. (3) B U T L E RJ,. .1.I-., Ttromos, 11. .\xi) 1 1 . x ~ ~ A S ?\Y.H . : J. Cheni. SOC.1933,674. (4) C o a x . E . J.! .%SI> E 1 ) s . t ~.J. ~ .T.: P w l e i t i s , .I tt itcids atid Peptides. Reinhold Publishing Corporation, S e w 1-oi-k (1043r. (5) EYRIXG, H . . ASLISTLIRS. .I.F;,: C'lieln. Iiev. 24, 253 (193'3). (61 H A R R I S11.. , l I I z E i d i . , I,. R . , A S U F O I . R TI,.: , Ind. Eng. C'hem. 34. 833 (1942). ( 7 ) JASSES. E . F.. XUSBROT, S.W.,.\xu R I E T ZE, . : Ind. Eng. Cheni., Anal. Ed. 16, 523 (1944). (8)J o s c s , C'. B., .\SI) ~ I E ~ H . uDI. ,IC.: . i w h . 13iochem. 3, 193 (1043). . J. G . : .J. Cheiu. Pliys. 2, 351 (1934). (9) H. P.:Textile Resixarch ,J. 15, 335 (l945i. (10) . T . : Ind. E n y . Chem.. Anal. E X . 5, 317 (1033). (11) (12) SC.~TCH.XRII. G . . .%xi)K I R K \ V ~ O,J,I )G, . : Phg-sik. %. 33, 297 (1932). (13) SH.~\\-. R . , . i s u RL-TLER,tJ, .1.I.,: P r o c . Roy. Soi,. (Idondon)A129, 519 (1030). (14) WLRD, W.H . , H I G H ,L . , .\xi) LL-SIIGRES. €I.P.: J .Polymer Research 1, 22 (1946).
