WIL~RED H. WARDAND JOHNJ. BAR~WLOVICH
Vol. 60
MOLECULAR KINETIC AND CHEMICAL PROPERTIES OF WOOL CORTICAL CELL FRACTIONS BY WILFRED H. WARDAND JOHN J. BARTULOVICH Western Utilization Research Branch, AgricuUural Research Service, United States Department of AgricuUure, Albany 10, Calijmia Receiued Februury 84# 1966
Wool corticals cells prepared by acid treatment have been resolved into two main fractions differing in density and composition. Proteins made soluble by reduction of wool exposed 96 hours to 6 N HC1 at 26’ were measured in 0.2 N NaCl plus 0.5 M mercaptoethanol a t pH 8. The molecular weight of protein from both fractions is about 40,000 and the molar frictional ratio 1.8 as calculated from the sedimentation constant 2.5 S and average intrinsic viscosity 0.19 deciliter per gram using the anhydrous prolate ellipsoid model. The relative ease with which the light fraction is released from the fiber, its lower sulfur content and lower density relate this fraction to the less consolidated “ortho” segment of the wool fiber. The heavy fraction then corresponds to the “para” segment. The sulfur content yield and intrinsic viscosity of separated cor-’-’ tical cell fractions change with time of treatment. The resulta indicate that differences in composition and physical properties are due to differences existing in the original wool although modified by acid treatment. The acid removes protein material relatively poor in sulfur and of low intrinsic viscosity from both cell fractions in such a way that the resistant residues approach similarity in sulfur content and increase in intrinsic viscosity. These results indicate that both wool fiber segmenta include resistant components of high sulfur content but differ in the proportion of components more readily dissolved by acid.
Introduction This paper is a report of research characterizing products of acid degradation of wool in order t o understand and control degradation during processing. The cortex making up the interior of an ordinary unmedullated wool fiber is its largest histological fraction, about 90% of the whole. A considerable variety of hydrolytic treatments has been found to break it up into spindle-shaped “cells” about 5 to 7 I.( wide and 80 L.I long. These have a fibrous structure. The surfaces have fine ridges parallel t o the long dimension and are covered with a chemically resistant layer. Reagents reported t o break up wool into spindle cells include concentrated aqueous ammonia, dilute potassium hydtoxide, concentrated sulfuric acid, dilute cetylsulfonic acid, pancreatin, trypsin and papain. Spindle cells have also been prepared by bacterial action. Binkley’ has prepared spindle cells by the action of G N hydrochloric acid near room temperature. The last reagent has been chosen for special study in this research. Much evidence shows that the process by which the cells are consolidated into a fiber is not uniform, so that one side of the filler nortnally differs from the other to a greater or lesser degree in chemical and mcchanical responses to cnviroiment. For example, a wool fiber cross-secttioncommonly shows bilateral differences in dye absorption, in natural pigmentation, in relative swelling by bases or acids, and in resistance to attack by enzymes and other chemicals including fresh bromine water and aqueous urea solutions with reducing agents. It has even been reported that wool-destroying insects prefer one side over the other. The bilateral asgtnmetry is closely related to fiber crimp. Since crimp and chemical stability are of the greatest imiiortance in processing and use of wool, bilateral differences are of practical as well as fundamental intprest. PAeparation of Cell Fractions for Molecular Kinetic Comparison.-An Idaho medium wool (\\’C-5)2 was used ( 1 ) C . H. Binkley, rinpriblished results from this Laboratorv. (2) This wool has been the object of other studics, for example.
W . H. Ward, C. H. Binkley and N. S . Snell. T e d . Research J . , 24, 314 (1955). whicli records its chemical composition.
for a molecular kinetic comparison of light and heavy cell proteins. The wool was Soxhlet-extracted with benzene and rinsed with hot water. A clean Fortion was soaked 96 hours at room temperature, about 26 , in 6 N hydrochloric acid. The acid:wool ratio wae 20:l. The wool was drained, rinsed carefully in distilled water, and beaten with water in a Waring Blendor.3 The separated cells were recovered by filtering on a hardened paper, washed until the washings gave no turbidity with silver nitrate, and kept in ethyl alcohol. On the basis of similar experiments the total yield of undissolved wool components is estimated to have been between 60 and 70%. The cells were fractionated in aqueous chloral hydrate density gradient columns4 and washed with water. The sulfur and nitrogen contents are given in Table I. The sulfur analyses indicate that the light fraction composed 43% of this preparation.
TABLE I SULFURA N D NITROGENCONTENTS OF MEDIUMWOOL, SPINDLECELL PREPARATION A N D FRACTIONS USED FOR MOLECULAR KINETIC STUDIES Material
Sulfur, %
Nitrogen, %
Original wool Spindle cell preparation Light fraction Heavy fraction
3.64 4.48 3.53 5.20
16.8 15.9 16.0 15.6
In view of the probable interaction between chloral hydrate and wool protein: chlorine contents of the cells are of interest. Typical preparations have been found to contain from less than 0.1 to 1.0% chlorine before fractionation and from 0.2 to 2.0% chlorine after contact with chloral hydrate. The latter figures correspond to 0.24 to 2.4% chloral not removed by washing. The higher values were observed with heavy fractions as noted in Table 111. For molecular kinetic studies reported here the cells were dissolved in 0.5 ill mercaptoethanol containing 0.2 ?tf sodium chloride and adjusted to pH 8. An undiwolved membranous residue amounted to 1 to 2y0 of the spindle cell weight. The amount of undissolved residue decreased with increasing time of exposure to acid. The heavy fraction gave about 10% more residue than the light. Each cell protein solution was dialyzed in commercial cellulose tubing against six time its volume of solvent. Tests of similar preparations Ahowed about 40y0 of the total nitrogen diffusible through the membrane. This proportion increased with longer exposure to acid but was not consistently different for the two cell fractions. The following measure(3) Mention of specific commercial products does not imply recommendation by the Department of Agriculture. (4) W. H. Ward and J. J. Bartulovich, T a t . Research J . , 28, 888 (1 955). (5) J. M. Preeton and M. V. Nimkar, J . TeililcInst.,ll,T446 (1950).
Sept., 1956
CHEMICAL PROPERTIES OF WOOLCORTICAL CELLFRACTIONS
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ments of intrinsic viscosity and Sedimentation constant of the dialyzed solutions then apply to material representing 35 to 40% of the original wool fiber, distributed roughly equally between light and heavy fractions. Molecular Kinetic Comparison of Cortical Cell Fractions. -Viscosities of the two solutions of cortical cell protein relative to the dialysates were measured a t 30" a t concentrations from 0.2 to 1.2%. Ubbelohde viscometers giving flow times of 300 to 500 seconds were used. The kinetic energy correction was negligible. Concentrations were found by difference from nitrogen analyses of the solution and dialysate. Measured densities of the solutions and dialysates were taken into account. Ultracentrifugal sedimentation constants were found for the same solution8 by means of a Spinco Model E centrifuge, using the synthetic boundary cell. Measurements were made a t the ambient temperature and referred t o water at 20" in the usual way. The intrinsic viscosity of the light fraction waa 0.20 deciliter per gram and the slope d(q.,/c)/dc 0.039 deciliter* gram-'. The intrinsic viscosity of the heavy fraction was 0.18 deciliter per gram and its slo e 0.035 deciliterP gram-*. These are least-squares values. h e sedimentation results showed no clear difference between the two preparations. The combined sedimentation results gave l / s = 0.40 O.O36c, where s is Svedberg units referred to water a t 20" and c is in grams of protein per 100 ml. The standard sedimentation constant at zero concentration is then 2.5 S.
in translating the intrinsic viscosity into the molar frictional ratio. Results calculated from some of these a9sumptions are given in Table 11. In the case of wool protein not exposed to acid but reduced and dissolved in aqueous urea, the prolate ellipsoidal model gives results most clearly agreeing with measurement of free diffusion. Provisional acceptance of the molecular weight and shape indicated by this model suggests the following conclusions. The molecular kinetic units of reduced protein from acid-prepared spindle cells are appreciably larger than those from the original wool reduced in aqueous urea. The spindle-cell protein forms a relatively elongated unit, but less so than the proteins solubilized in urea. The mean molecular weights of proteins from the different spindle cell fractions may differ slightly, but probably not more than 10 to 15%. Such a difference would permit an actual difference of as much as 0.4 S in the sedimentation constants or 0.05 deciliter per gram in the intrinsic viscosities. Such differences approximate the greatest observed in these studies. Electrophoretic Analysis of Cortical Cell Protein .-The portion of the wool fiber more resistant to chemical attack has been found repeatedly (for example by Lindley.6 by Binkley,2 and by Mercer and colleagues7~*) to differ in composition from the less resistant part. In several instances the resistant part has been identified with the para-cortex. The composition of the ortho segment has been estimated from the difference in composition of the whole wool and its resistant portion.8 Thus, evident differences in the contents TABLE I1 of acid and amino acids have been ascribed directly to the different segments. This inference may be invalid because MOLECULAR PROPERTIES OF REDUCED SPINDLECELLPRO- of the presence of other wool fiber components in the disTEIN FROM VISCOSITY AND SEDIMENTATION solved portion, including material from the resistant segBaais of determination //lo M ment. However, if the two cortical segments do differ in Solute taken as unsolvated hard SDhereS 1 17.500 their contents of ionizable groups this fact should be readily Prolate ellipsoids of revolution, uniolvatetl 1.81 42; 600 shown by electrophoresis. Whole and fractionated cortical cell preparations were Oblatee~Psoidsofrevolution,unsolvated 2 . l 6 55,600 therefore tested by electrophoresis on paper (Whatman no. Solvated sphere, the bound solvent e q u d 3), with xylose and Armour bovine plasma albumin as reference substances. An unfractionated cell preparation in composition and density to the origifrom Idaho Rambouillet (WC-4) wool exposed to 6 N hydronal solvent 2 , 19 56, chloric acid for 110 hours a t room temperature was used for Solvated sphere, water alone bound, with exploratory work, supplemented by tests with light and density of normal water 3.47 113,000 heavy fractions from the same wool treated 66 hours at 30".
+
TABLE 111 PROPERTIES OF CORTICAL CELLPREPARATIONS FRACTIONATED AFTER VARIOUS TIMES OF ACIDTREATMENT AT 30" Time of treatment, hr. 28 48 66 90 Fraction Light Heavy Light Heavy Light' Heavy Light" Heavy Yield, yooriginal wool 38.2 25.5 27.9 11.9 14.4 6.8 1.2 10.8 Yield, % cortical cell preparation 60 40 70 30 68 32 10 90 Total sulfur, % 4.19 5.24 6.46 7.35 6.34 6.27 6 80 6.68 Cystine sulfur, % 4.0 4.8 4.8 5.3 5.8 5.9 6 5 5.9 Nitrogen, %) 15.6 15.3 15.3 15.2 14.7 14.5 14.4 14.5 Chlorine, % 0.51 2.04 0.25 0.40 0 32 0.36 0.28 0.22 Densityb, g. cm.-* 1.482-1.515 1.489-1.500 Range" Separate layers 1.450-2 1 . 476-gd 1.452 1.475-9 Intrinsic viscosity, dl./g. 0.147 0.115 0.228 0.158 " Note that these "light" fractions do not correspond in density or sulfur content to those isolated after shorter treatments. * Densities were measured in a ueous chloral hydrate gradients at 30" by comparing positions of cortical cell material with those of standardized floats. % eh"! 66 and 90 hour preparations did not give separate layers in gravity separation, but were fractionated in the centrifuge in the usual way. An intermediate layer of density 1.468-1.475 can be distinguished just above this heavy fraction. Molecular Weight and Molar Frictional Ratio.-Estimates of the molecular weight and molar frictional ratio of the reduced spindle cell protein not diffusing through cellulose membrane were made from the mean intrinsic viscosity 0.192 deciliter per gram, the standard sedimentation constant 2.49 S and the assumed value of the partial specific volume 0.727 estimated from the amino acid composition of whole wool. The sedimentation constant and partial specific volume permit calculation of a minimum possible molecular weight, 17,500 taking the molar frictional ratio t o be unity. The actual molecular weight is this minimum value multiplied by the 3/2's power of the molar frictional ratio, which is found from the intrinsic Viscosity and partial specific volume. Several ditrerent models may be assumed
The properties of these fractions are included in Table 111. Analyses in 0.1 M mercaptoethanol with 0.05 M NaeHPOl (pH 8.1) or 0.1 N NaCl (pH adjusted to 8.6) or in 0.05 M Na2S08 (pH 9.38) in each instance showed a single component moving slightly faster than the plasma albumin. The components were located by oven-drying in the presence of aniline vapor (to show the x lose) followed by staining with 0.1% bromphenol blue in 5& mercuric chloride and 5% (6) H. Lindley, Nafws, 160, 190 (1947). (7) E. H. Mercer, R. L. Golden and E. E. Jeffries, Tezt. Research J.. '24, 615 (1954). (8) R. L. Golden, J. C. Whitwell and E. H. Mercer, %%id.,46, 334 (1955).
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WILFREDH. WARDAND JOHN J. BARTULOVICH
acetic acid. Part of the unfractionated sample was treated with 1.6% peracetic acid, freshly diluted from Becco 40% acid, for 20 minutes a t room temperature, washed free of oxidant, and extracted with 0.05 N ammonia. This extract showed a single component slightly slower than the unoxidized material. These tests give no indication of any differences in electrophoretic properties of different spindle cell fractions. The different fractions of this preparation, therefore, do not differ appreciably in their net amounts of groups ionized a t pH 8 to 9. Results to be summarized in the next section suggest that one fraction disappears or merges its identity with the other as the time of treatment is extended. It is therefore possible that further study of fractions from wool given the shortest possible treatment might show additional electrophoretic components. Changes in the Insoluble Wool Residue with Time of Exposure to Acid.-In order to clarify the relations of the light and heavy cortical cell fractions to the original fiber, preparations were made with different times of acid treatment. Trends in relative and absolute yield, density and sulfur, cystine and nitrogen contents were established. For this series Idaho Rambouillet (WC-4) wool was treated with 6.1 N hydrochloric acid at 30.0'. The wool disintegrated appreciably more quickly than a t room temperature. The minimum time for substantial disintegration a t 30" was about 30 hours as opposed to about 90 hours a t room temperature. The results are given in Table 111.
Vol. 60
From these results, the light cortical fraction is estimated to amount to about 60% of this original wool. This figure is indicated both by the relative yields as isolated and by extrapolating backward to zero time of treatment. In addition, the disappearance of the less dense fraction with extended treatment, the increase in the density of the material isolated, the over-all increase in sulfur and cystine content confirming observations of Lindley,6 the increases in intrinsic viscosity of the reduced protein fraction, and the failure to find appreciable differences in electrophoretic properties and ultracentrifugal sedimentation of these protein fractions suggest very strongly that both cortical cell fractions include protein components of high sulfur content that are considerably more resistant to degradation by acid than the rest of the fiber. The difference between the two cortical segments then includes differences in proportions of components with lower sulfur that are more readily dissolved by acid.
Acknowledgments.-Elementary analyses reported are the work of L. M. White, G. E. Secor and A. M. Mylne. Cystine was determined by J. E. Moore. J. W. Pence helped us in carrying out paper electrophoresis. We are likewise indebted t o H. P. Lundgren and other eo-workers both in this Laboratory and elsewhere for many useful suggestions for this and further research.
