ARTIFICIAL FI
By procedures developed in this laboratory in accordance with this principle, it is possible to prepare highly oriented fibers from fibrous and corpuscular proteins. For example, fibers that possess tensile strengths and water iesistance comparable with those of natural protein fibers have been prepared from egg albumin and from chickenfeather keratin. These commodities serve as typical examples of corpuscular and fibrous proteins and, in addition, are available commercially in significant quantities. Recent statistics show that over 170,000,000 pounds of chicken feathers are available annually and over 26,000,000 pounds of inedible technical egg white, much of which goes to waste with the eggshells in the course of processing. The technique has been tested and found applicable to certain other technical proteins. The object of the present paper is to outline the method and to illustrate the principles involved. The use of detergents for dissolving is not new. In - moteins fact, a considerable number of patents have been granted on empirical mixtures of proteins with detergents, and these mixtures have applicptions as adhesives, sizes, finishes, soaps, and the like. Until recently, however, little was known concerning the nature of protein and detergent in mixtures.
from
Corpusc
Fibrous
oteins
HAROLD P. LUNDGREN ,LND RICH-4RD A. O’CONNELL
I
Western Regional Research Laboratory,
U. S. Department of Agriculture, Albany, Calif.
INTERACTION
I
NTEREST in the formation of artificial fibers from proteilih has been stimulated by the war emergency, as well as by favorable developments since Ferretti, in 1935, showed that usable protein fibers could be made from casein. Fibers from a variety of proteins, including casein and those from soybeans, certain vegetables, and fish, have been described, but so far no fiber has been reported with qualities comparable to those of such natural protein fibers as silk and wool. As the work of Astbury, Mark, Meyer, and others (8, 3, 4,7 , 11, iW, 13, 18, 19,$0)has established, the structural unit of natural fibers is a chain molecule. Such units, when not completely cross-linked, are capable of being curled because of free rotation .around single bonds, and this characteristic is necessary for flexibility in fibers; they are also capable of being oriented and t h u s the maximum opportunity is afforded for cross linking .and for the formation of crystal lattice structure, which favors fiber strength. Theoretically all known proteins are structurally adaptable to fiber formation. They consist either of open polypeptide chains appropriate for alignment into oriented fibers or of folded chains capable of being unfolded. It should be possible, therefore, to transform either type of protein into desirable fibers. Work along this line has been done by Astbury and his associates, -who attempted to use corpuscular proteins dispersed in aqueous urea solutions (6) in preference to the caustic soda commonly used in making commercial protein fibers. There is no evidence that the use of urea solutions, with or without added spinning .“auxiliaries”, has proved practical; neither have the fibers prepared by this procedure been reported as having high molecular orientation or strength. T o take advantage of the structural adaptabilities of proteins for the development of fibers, i t has been found practicable to employ certain synthetic detergents (14). These agents act in several ways: They are excellent solvents for proteins and cause little, if any, degradation of the chains; they unfold corpuscular proteins by breaking the bonds which hold the chains in this configuration; and they form complexes with both fibrous and corpuscular or globular proteins which are easily precipitable into a plastic state favorable for drawing into fibers. Finally, following the formation of the fiber, the detergent can be removed and recovered. The protein chains are left in a state in which they can be drawn into alignment, and new secondary or primary valence cross linkages can be formed that increase the strength of the fiber.
IN PROTEIN-DETERGENT MIXTURES
Evidence of structural changes accompanying the solution of corpuscular proteins in detergents was shown by Anson ( 1 ) who demonstrated that denaturation occurs and that detergents differ in their ability to cause such change. I n a study of the nature of the interaction between proteins and detergents in this laboratory, it was observed that, when certain corpuscular proteins are dissolved in aqueous solution of some detergents, the viscosity begins t o rise immediately and after a time it approaches a maximum which depends upon the conditions. It has been found convenient t o compare maximum viscosity reached by mixtures of varying ratios. Figure 1shows such a comparison for mixtures of 4% crystalline native egg albumin and sodium alkyl Two benzenesulfonate solutions which have stood 24 hours. maxima in viscosity are seen. In solution where the total concentration of solids is above 15% and where the proportion of de-
Proteins in general are potential fiber-building material. By proper manipulation of proteins derived from surplus and waste agricultural and industrial commodities, fibers have been made with molecular orientation, strength, and moisture-absorbing characteristics comparable with those of natural protein fibers. These fibers have been made with dispersing agents which are mild in comparison with those commonly used for commercial protein fibers. The agent (a detergent) used not only serves as a solvent for proteins but also interacts in appropriate mixing proportions with proteins to form complexes containing unfolded protein. Solutions containing complexes of appropriate composition can be used to make fibers, either by passage through a spinneret into a coagulating bath of salt solution, or by precipitation with inorganic salt with subsequent drawing of the precipitate into a fiber. The detergent can be recovered with aqueous acetone, leaving the peptide chains in the resulting protein fibers in such a state that they can be oriented by drawing in live steam. Secondary and primary valence cross linkages then occur which hold the peptide chains in a highly oriented condition when the fiber is removed from the steam bath. As the degree of orientation in the fibers is increased, tensile strength and water resistance increase although the range of elastic deformation decreases.
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INDUSTRIAL AND ENGINEERING CHEMISTRY
tergent present is greater than 1 part t o 3 parts of albumin, the change in viscosity can be followed by the accompanying development of flow birefringence. Neither egg albumin nor an alkyl benzenesulfonate solution alone shows this effect under these conditions. The appearance of flow birefringence, which in this case is positive in sign, can be shown t o Figure 1. Relative Viscosities of Mixtures of Proteins in Sodium be characteristic of Alkyl Benzenesulfonate the manifestation of The egg albumindetergent mixtwere r o d s h a p e d r a t her at pH 6.5 and were pre ared in 4% molutions of the protein anJdeter ent. in 1 0 than of plate-shaped buffer salt. The chicken-featter kerat% solutions were at pH 12.0 and were preparticles, and can be pared with 4% protein and detergent. All accomted for by the measurements were made by the ca illary .method after the mixtures were afiowed unfolding of the proto stand at least 24 hours. tein peptide chains. This is compatible with current ideas regarding protein structure. After standing for some time the more concentrated mixtures showing these properties set t o gels which are birefringent on deformation. This behavior is likewise consistent with the manifestations of extended chain molecules. I n contrast t o native egg albumin, solutions of reduced chicken-feather keratin in alkyl benzenesulfonate show birefringence immediately on preparation and exhibit no significant change in viscosity with time. As Figure 1shows, when the ratio of de tions is increased, except fo ratio of protein t o detergent mixtures falls steadily in pr
371
tional to the relative amounts of protein and detergent present in the complex. The latter is determined by the mixing proportions. Analysis of the electrophoretic patterns shows that, when the sulfonate is added to an excess of native protein, a complex forms that possesses a constant ratio of protein t o detergent, the free protein appearing as a slower, separate boundary. Also, when excess detergent is added to the protein, two boundaries appear. I n this case the slower boundary represents a complex and the other the free detergent. In the mixture containing excess protein, the composition of the complex is approximately constant, with 3 parts by weight of protein to 1 of detergent, Where free detergent is present, analysis of the complex is more difficult, but its composition appears to approach a ratio of approximately 2 parts by weight of detergent t o 1 of protein. In the middle region, where no free detergent or free protein is present, the complexes combine in the proportion in which they are mixed. In contrast to the behavior of mixtures of native proteins and detergent, solutions consisting of a high proportion of heat-denatured egg albumin t o detergent exhibit no free boundary in electrophoretic analysis. Neither is there an increase in viscosity to a maximum under this condition. Except for this absence of B lutions having a high proportion of protein t o detergent, the electrophoretic behavior of heat-denatured egg albumin when mixed with the sulfonate is similar t o that of the native albumin. These results have been confirmed, where possible, by chemical analyses of the complexes precipitated from solution by electrolyte. The results of such analyses appear as squares in Figure 4. It is seen that the complexes possess fairly constant composition of approximately 70 parts by weight of protein to 30 of detergent ______f
ASCENDINQ BOUNDARIES
-
DESCENDINQ BOUNDARIEB
3 2 1 1 2 3 80-10 native proteih-detergent. Boundaries: 1, protein-detergent complex; 2, excess native protein; 3, delta and epsilon boundaries.
.. . , .._. 2 1 1 2 60-40 native proteindetergent. Boundaries: 1, protein-detergent complex; 2, delta and epsilon boundaries.
*
is peculiar t o proteins having a f X-ray patterns (23)of the of the complex containing mo
3 2 1 10-90 native protein-detergent.
1 2 3 Boundaries: 1, free detergent;
2, protein-detergent complex; 3, delta and epsilon boundaries.
1 1 90-10 heat-desatured proteindeterpent. Boundaries: 1, proteindetergent complex; de ta and epsilon boundaries were too small to be seen.
Figure 2.
tergent (16). Figure 3 shows that the rates of migration of these complexes-that is, their magnitudes of charge-are propor-
Electrophoretic Diagrams of Egg AlbuminAlkyl Benzenesulfonate Mixtures
The total protein plus detergent concentration was 1%; pH = 6.58 = 0.1; t = 0.75" C.
I N D U S T R I A L A N D E N G I N E E R I N G CHEMISTRY
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20
40
100
80
60
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ao
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40 DETERGENT
20
0
ALBUMIN
Relation of Mobility of Protein-Detergent Complex to Composition
Figure 3.
0 Mobility ws.
mixing ratio
0 Mobility us. composition
A Mobility of denatured albumin-
detergent complexee US. composition The solid circles on the vertical line at approximately 75% rotein represents complexes of incompletely denatured protein. gobility of alkyl benaenesulfonate, -20 X 10-5, is shown at the extreme upper left; mobilities of native albumin, -5.6 X 10-6. and heatdenatured albumin, -6.7 X 10-5, are shown below and above, respectively, at the lower right. The same scale is used for mixing retio and for complex composition.
(by difference) as long as there is 70c0or more albumin in the mixture. Below 70% albumin in the mixture the composition of the complex varies in the same proportion as the mixing ratio. Since the precipitates from the solutions having a mixing ratio of greater than 70-30 detergent-protein were very slimy and difficult t o separate, no values have been obtained for this region. It becomes apparent that the complex having approximately constant composition formed in mixtures containing, roughly, more than three parts of native protein t o one of the detergent represents a definite combining capacity of the native protein for the detergent. A similar limit is approached in the case of excess free detergent which is the same whether native or denatured protein is used. The latter limit represents a n upper level for the
E 100 A
L
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/A’
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IL
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Figure 4. Relation between Composition of ProteinDetergent Complexes and Mixing Ratio 0 Calculated from electrophoresis data on native albumin-detergent mixtures 0 Calculated from nitrogen analyses of precipitated complex A Calculated from electrophoresis patterns of denatured albumindetergent mixtures
Vol. 36, No. 4
combination of denatured protein for detergent. It is significant that these limits in combining capacity correspond with the maxima found in the viscosity measurements mentioned earlier, The diffusion constants for mixtures of egg albumin and sodium alkyl benzenesulfonate are always lower than those for either constituent alone. I n the absence of ultracentrifugal sedimentation data, it is impossible t o decide whether this is due to increase in mass of the kinetic units, to a change in symmetry, or to a combination of both. However, if we use the diffusion constant of the detergent and assume (10, 16, 17) that the micelles are spherical, we can obtain a calculated micellar weight of approximately 15,000 for the detergent. With 45,000 as the molecular weight of egg albumin, the proportions, mentioned above, for the limiting combining capacities of native and denatured egg slbumin amount to one molecule of native protein with the equivalent of one micellar unit of the detergent, and one molecule of denatured protein with six units of the detergent. The increase in the ability of the detergent to unite with protein beyond the mixing ratio of 75-25 protein-detergent is ascribed to the liberation of new points of attack due to structural unfolding. The results reported here were obtained with egg albumin and sodium alkyl benzenesulfonate, but there is evidence that other proteins and other detergents react similarly. Recently Miller and Andersson ($1) described the behavior of mixtures of insulin and alkyl sulfate, They showed by ultracentrifugal analysis and diffusion measurements that a complex forms which corresponds to the union of half a molecule of insulin with one micelle of detergent. FIBER FORMATION FROM PROTEIN-DETERGENT COMPLEXES
When precipitated by salts the protein-detergent complexes will be either flocculent or slimy, depending on the ratio of protein to detergent. I n the region where the proportion of protein is high, the precipitates are flocculent and the particles do not adhere. Beyond the ratio of about 75-25 protein-detergent,
Figure 5. Fiber Spinning. The Fibers Form When the Viscous Prot e i n-De t e r g e n t Sirup Strikes the Fluid in the Bath and Coagulates
April, 1944
INDUSTRIAL AND ENGINEERING CHEMISTRY
where the combination is no longer in constant proportion, where increase of birefringence becomes evident, and hence where unfolding of the corpuscular protein apparently occurs, the precipitates become increasingly slimy as the proportion of detergent increase. If the precipitates are not too slimy, they can be drawn by hand into fibers. In the case of egg albumin and chickenfeather keratin disflersions with alkyl benzenesulfonate, 50.2 'C. 100%. ductile precipitates are formed when the mixing ratio lies between 40-60 and 60-40 protein-detergent. ViSCOUS solutions of mixturev having these proportions a n d compositions above 17% of total solids I I I I can be extruded I 2 3 4 through the holes of HOURS EXTRACTION a spinneret (e.g., Figure 6. Extraction of Alkyl Benwith one hundred zenesulfonate from Egg Albumin holes, each 0.003 Fiber with 60% Aqueous Acetone inch in diameter) into a coagulating bath of salt solution to give fibers that can be stretched and reeled (Figure 5). When fibers prepared in this manner are allowed to dry, they are weak and take up water readily. Attempts t o produce orientation by drawing the fibers in the presence of steam failed. At best, an elongation of 100% was reached, and the resulting fibers were brittle and not very strong.
373
with the increase in orientation as e result of streching, there is no corresponding increase in tensile strength. Elongation in Steam, % ' 100 200 300
Av. Tensile Strength, Lb./Sq. In. 18,000 25,000 28,000 38,000
400
Fibers have been prepared from chicken-feather keratin and egg albumin possessing tensile strengths of more than 70,000 pounds per square inch. These values compare favorably with those of natural fibers: Fiber Flax (9) Ramie (9) Nvlon Cotton ()'(8) Viscose rayon Acetate rayon
Breaking Stren t h 1000 fb.) Sq. In. Up to 166 129-135 72-100 40-111 31-88
23-110
Breaking Strength
Fiber Silk (9) Tech. egg albumin Chicken-feather keratin WOO](9)
Commercial ossein Commercial soybean
1000 Lb.)
Sq. In. 46-74 20-70 UD to 80 i7-25 up to 10 up to 10
Along with the increase in orientation and tensile strength, there is a corresponding increase in water resistance. Even though they have not been fixed, the fibers with a high degree of molecular orientation show moisture retention comparable t o that of wool, as is shown in the table which follows on the next page.
REGENERATION OF PROTEIN
The detergent can be recovered from the fiber with certain solvents, such as aqueous acetone. Figure 6 shows the course of extraction of detergent from fibers by the use of an excess of 60% aqueous acetone, As the following table shows, higher or lower concentrations are less effective: Original albumin-alkyl benzenesulfonate fiber After 48-hr. extn. a t 26O C. with 3 ohanges of: 20% aqueous acetone 40% aqueous acetone 60% aqueoua aoetone 80% aqueou8 acetone 100% acetone
Protein, % 50
Figure 7. Regenerated and Stretched Fibers from Technical Egg White
75 86 99
78 58
ORIENTATION O F REGENERATED FIBERS
Following the removal of detergent it is possible to draw the fibers over 3oOy0 in live steam. I n some cases an extension of over 700% has been reached. After proper annealing a t lower temperatures, such fibers (Figures 7, 8, and 9) exhibit positive birefringence (Figure 10) and a high degree of molecular orientation as determined by x-ray diffraction analysis (Figure 11). Detailed study of the x-ray patterns by Palmer (93) of this laboratory has shown that the distances, calculated from the diffraction patterns obtained from the fibers, correspond t o those of p-keratin, a typical fibrous protein. Egg albumin fibers were equilibrated 24 hours a t 70" F. and 65% relative humidity, Measurements made on the Scott inclined-plane tester show that,
Figure 8. Thread and Filament Prepared from Technical Egg White (X 150) Left. Polyfilament thread, (I u n mechanically and containing PO0 filaments fused together; right, single filament drawn by hand.
Figure 9. Polyfilament Threads of Fused Filaments Spun from Reduced Chicken-Feather Keratin Dispersed in Aqueous Alkyl Benzenesulfonate (X 150) Left. Unbleached from keratin derived from red ' feathers; right, bleached.
INDUSTRIAL AND ENGINEERING CHEMISTRY
314
Figure 10. Optical Birefringence of Polyfilament Thread of Egg 4lbumin, Showing a High Degree Of Orientation of Structural tinits
__I .i Fiber
Figure 11. X-Ray Diagrams of Fibers Prepared from Recrystallized Egg Albumin L e f t . Unstretched unoriented pattern. Right. Stretched (400%) oriented pattern; calculated distances correspond to those of @-keratin characterietir of silk or stretched wool.
ergeiitmay involve long-range forces between the ionic groups and short-range forces (van der Waals) between the exposed nonpolar groups of the protein and of the detergent ($$, 2-4). It is also possible that detergent may become attached through van der Waals forces to the nonpolar portion of detergent ions a,lrea,dy bound ionicallv in limited amounts by the protein. ACKNOWLEDGMENT
The photomicrographs were made by R. M. Reeve of this laboratory. D. W. Elam assisted with the viscosity measurements. The solutions of reduced chicken-feather keratin were furnished by C. B. Jones of this laboratory. The authors wish to’express their appreciation to M. J. Blish and to G. H . Brother for their interest in this investigation, and t o acknowledge the mechanical services of John R . Hoffman and E. I,.Muller in the construction of the spinning equipment,. LITERATURE CITED
Anson, 31. L., J . Gen. Physiol., 23, 239-61 (1939). ( 2 ) Astbury, W. T., “Advances in Enzymology”, No. 3, pp. 63-105, New York, Interscience Pub., 1943. 13) Astburv. W.T.. Chemistrv & Industru. 19.491-7 (1941). (4) Astbury, W7.T., “Fundamentals of Fibre Structk-e”,’ London, Oxford Univ. Press, 1933. ( 5 ) Astbury, W. T., nickinson, S., and Bailey, K., Biochem. J . , 29, (1)
2351-60 (1935).
(0)Baker, W. 0.. Fuller, C. S., and Paee. N. R., J . Am. Chem. Soc., 64, 776-82 (1942)
Frey-Wyssling, A., ProtopZasma, 25, 261-300 (1936). (8) Harris, M., Bur. Standards J. &search, 10, 475-8 (1933). ENQ.CHEM.,34, (9) Harris, M., Mizell, L. R., and Fourt, L., IND. (7)
833-8 (1942).
(10) Hartley, G. S., Kolloid-Z., 88, 22-40 (1939).
(11) Kratks, O., Ibid., 64, 213-22 (1933). (12) I b i d . , 68, 347-50 (1934). (13) I b i d . , 70, 14-19 (1935). (14) Lundnren. H. P.. J . Am. Chem. Soc.. 63. 2854-5 (1941). (15)
Lundgren, H. P:, Elam, D. W., and O”Connel1,R . A., J . Bid.
(16)
McBain, 3. W., “Advances in Colloid Science”, Val. I, pp. 99142, New York, Interscience Pub., 1942. McBain, J. W., Nature, 145, 702-3 (1940). Mark, H., IND.ENQ.CHEiv., 34, 449-54 (1942). Meyer, K. H., “Natural and Synthetic High Polymers”, New York, Interscience Pub., 1942. Meyer, X. H., and Mark, H., Ber., 61, 593-614 (1928). Miller, G. L., and Andersson, K. J., J . Biol. Chem., 144, 475-86
Chem., 149, 183-93 (1943). (17) (18) (19) (20) (21)
(1942).
(22) Palmer, K. J., J . Phys. Chem., 48, 12-40 (1944). (23) Palmer, K. J., and Galvin, J. A , , J . Am. Chem., Soc., 65,2187-90 (1943) (24)
Steinhardt, J., Fugitt, C. H., and Harris, M., J . Research Natl.
Bur. Standards, 28, 201-16 (1942). (25) Wilson, R . E , , and Fuwa. T., IND.ENQ. CHEM.,14, 913-18 (1 922;,
