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J. D. LEEDERAND I. C. WATT
The Role of Amino Groups in Water Absorption by Keratin
by J. D. Leeder Division of Textile Industry, C.S.I.R.O. Wool Research Laboratories, Belmont, Victoria, Australia
and I. C. Watt Division of Textile Physics, C.S.I.R.O.Wool Research Laboratories, Ryde, New South Wales, Australia (Receioed February 16, 1966)
The effect of amino groups in keratin on the keratin-water isotherm has been investigated by measuring isotherms of a series of partially deaminated wools. A plot of residual amino group content against equilibrium water content is linear a t humidities up to 80% relative humidity. Extrapolation of this plot to zero amino group content enables an isotherm to be constructed for wool containing no amino groups. The reduction in water content can be expressed as a Langmuir isotherm a t relative humidities below 25Oj,. At higher humidities the absorption of a second and third water molecule on each amino group causes deviations from the Langmuir isotherm and variations in the over-all energy of attachment of water molecules with change of water content. Above approximately 80% relative humidity additional absorption of water on water within the wool also occurs.
Introduction It is generally accepted that proteins absorb water vapor by binding water molecules to hydrophilic sites a t low relative humidities, followed by condensation or multimolecular absorption as the humidity increases. 1-4 At present no clear-cut distinction can be made between bound water and mobile or condensed water, the relative proportions of each varying with the total moisture content of the ~ y s t e m . ~ Chemical and physical modifications of keratin have been related to changes in shape of the keratinwater isotherm.6 Changes in the polar nature of wool result in variation of water content a t low relative humidities, while physical changes which modify the forces opposing swelling of the keratin network result in deviations from the normal isotherm a t high humidities, ie., in the solution region proposed by Katz.' For the keratin-water system the solution region has been defined as the SO-lOO% relative humidity range.* The main polar groups in proteins are the free amino, carboxyl, and hydroxyl groups of the amino acid side chains. Mellon, et aZ.,* found that benzoylation of amino groups in casein reduced the water content, particularly a t low relative humidities, while Kanagy and Casselg obtained similar results with collagen after The Journal of Physieal Chemistry
deamination, acetylation, and esterification treatments. When applied to wool, deaminationlO was reported to give no change in the isotherm. However, Watt and Leeder6 have shown that acetylation and esterification result in significant reductions in water content a t low humidities although increased water uptake may occur a t high humidities owing to structural changes brought about by the chemical reaction. Modification of the hydrophilic properties of wool by chemical reactions which introduce new groups can give effects other than the desired specific reaction. For instance, Leeder and Lipson" found that acetyla(1) A. B. D.Cassie, Trans. Faraday SOC.,41, 450,458 (1945). (2) L. Pauling, J . Am. Chem. SOC.,67, 555 (1945). (3) A. D.McLaren and J. W. Rowen, J . Polymer Sei., 7,289 (1951). (4) J. J. Windle, ibid., 21, 103 (1956). (5) M.Feughelman and A. R. Haly, Teztile Res. J . , 32, 966 (1962). (6)I. C. Watt and J. D. Leeder, Trans. Faraday Soc., 60, 1335 (1964). (7) J. R. Kate, ibid., 29, 279 (1933). (8) E. F. Mellon, A. H. Korn, and S. R. Hoover, J . Am. Chem. Soe., 69, 827 (1947). (9) J. R. KanagV and J. M. Cassel, J . Am. Leather Chemists' Assoc., 52, 248 (1957). (10)J. B.Speakman, J . SOC.Chem. Ind. (London), 49, T209 (1930). (11) J. D. Leeder and M. Lipson, J . AppE. Polymer Sei., 7 , 2053 (1963).
ROLEOF AMINOGROUPSIN WATERABSORPTION BY KERATIN
tion and esterification treatments alter the density of wool such that sorption properties, particularly rate of absorption, are changed. The new groups may be hydrophilic to an unknown extent, so it would be advantageous to investigate the effect of polar groups on the keratin-water isotherm by using reactions designed to remove these polar groups, without leaving bulky substituents in their place. The attachment of water to amino groups in proteins occurs with an energy of binding higher than the energy needed to attach to other hydrophilic groups12so modification of this group should have a greater effect than modification of other hydrophilic sorption sites. The effect of deamination on water sorption by keratin is reported in this paper. Experimental Section Absorption isotherms were measured a t 35" as previously described,13using a quartz spiral spring balance. The wool was from the same source as that used in earlier work6 and was given a similar cleaning procedure. Deamination Treatments.14 Deamination was carried out with van Slyke reagent as follows. Samples (5 g.) of wool were treated with 165 ml. of 25.6% aqueous sodium nitrite 35.3 ml. of glacial acetic acid a t 20" for (a) 1 day, (b) 2 days, (c) 4 days, (d) 8 days, and (e) 16 days, with daily changes of reagent. Treatments d and e were washed with water and carded on a small mechanical carding machine every 4 days to assist in obtaining even and exhaustive treatment. The Orange 11-formic acid dye uptake method of llaclaren16 was used in the estimation of total basic group content. Table I contains the results of analyses for residual basic groups in the deaminated wools.
+
Table I : Basic Groups Analysis of Deaminated Wools Total basic groups, mequiv./g.
Treatment
Untreated Treatment Treatment Treatment Treatment Treatment
a b c d e
0.83 0.49 0.42 0 .'34 0.17 0.10
For the purpose of the present work, it will be assumed that deamination only modifies amino groups. Amino acid analyses (kindly carried out by Mr. A. S. Inglis) indicate reaction with tyrosine, butdiazotization
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of tyrosine occurs in the ortho position,16probably leaving the phenolic hydroxyl unchanged as a water sorptionsite. Results and Discussion Comparison of the activation energies of diffusion and heats of hydration for the wool-water system with the values obtained for the individual polar groups ~ ~ Speakman12 to show that enabled Watt, et U Z . , ~ ' ~ and initial uptake of water vapor by dry wool is most likely to occur on the polar amino, carboxyl, and hydroxyl groups. The heats of hydration for these groups have been set at 16.8 kcal./mole for NH3+groups, 7.4 kcal./ mole for COO- groups, and 5.7 kcal./mole for OH groups,'2 while the activation energy of drying of wool increases from approximately 5 kcal./mole a t 5% water content to approximately 16 kcal./mole a t dryness.'' This suggests that at low relative humidities water is first sorbed onto amino groups, then onto carboxyl and hydroxyl groups as the water content of the system increases. Removal or inactivation of these polar groups would therefore be expected to have a profound effect on the wool-water isotherm, particularly in the low humidity region. The curves in Figure 1 compare the water vapor isotherms for the three deaminated wool samples a, c, and e with that for untreated wool. The isotherms are considerably modified by deamination, but the reduction in water content is not proportional over the entire isotherm. For treatment e the equilibrium water content (e.w.c.) is reduced by approximately 30% a t low humidities, while at 80% relative humidity the reduction is of the order of 17%. These changes are contrary to those observed by Speakman,l0 probably owing to the more severe deamination conditions used in the present study since increased severity of treatment results in greater reductions in water content. It is possible that conformational changes in the keratin structure occurred during the deamination treatment as indicated by the increased water uptake in the solution region of the isotherms. Any effect on the wool-water isotherm owing to degradative action will be restricted to the solution (12) J. B. Speakman, Trans. Faraday Soc., 40,6 (1944). (13) I. C. Watt, Teztile Res. J.,32, 1035 (1962). (14) G.H.Elliott and J. B. Speakman. J. SOC.Dyers CO~OUTiStS,59, 185 (1943). (15) J. A. Maclaren, Arch. Biochem. Biophys., 86, 175 (1960). (16) J. S. L. Philpot and P. A. Small, Biochem. J . , 32, 542 (1938). (17) I. C.Watt, R. H. Kennett, and J. F. P. James, Teztile Res. J., 29, 975 (1959). (18) I. C . Watt, ibid., 30,443 (1960).
Volume 69,Number 10 Odober 1966
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J. D. LEEDERAND I. C. WATT
35r
PERCENTAGE
RELATIVE
HUMlDlPl
Figure 1. Water absorption isotherms a t 35": curve A, treatment a; curve B, treatment c; curve C, treatment e; - - - -, unmodified wool.
region of the isotherm,6 ie., above 80% relative humidity. The curves in Figure 1 indicate less water uptake in the solution region as the severity of treatment increases despite an expected increase in degradation. However, the reduced water content and consequent reduction in swelling of the fibers delays the onset of the solution region and decreases the water uptake a t high humidities. Deamination with the van Slyke reagent converts some amino groups t o hydroxyl g r o u p ~ , ~soJ ~not all the amino groups will be converted to nonpolar residues. However, Valentinelg has shown that hydroxyl groups have very much lower sorbing powers than amino groups at 65% relative humidity, so any effect of new hydroxyl groups will be of secondary importance a t this stage and will not affect the general conclusions of the present paper. From the sorption isotherm data for the treated wools and the corresponding amino group analyses from Table I, values of equilibrium water contents have been taken a t selected relative humidities and plotted against the number of residual basic groups in the for benzoylated casein manner used by Mellon, et d.,* (see Figure 2). The relationship in each case is linear except a t the highest humidities, where structural modifications affect the water content. This linear relationship is significant since it demonstrates that even at low humidities all the amino groups are equally accessible to water vapor despite the complex morphological structure of wool keratin. Extrapolation to zero basic group content a t each relative humidity enables the isotherm shown as curve B of Figure 3 to be constructed. The difference beThe Journal of Physical Chemistry
I
0
I
.2
I
.4
I
.6
I
1
1.0
.8
TOTAL BASIC GROUPS (Mcqlg.) Figure 2. Equilibrium water content a t 35" us. residual amino group content, a t various relative humidities.
35r
/
//
--I
!% 20
w K
k
3 15-
1
0
40 PERCENTAGE RELATIVE
80
20
100
HIIMI[My
Figure 3. Water absorption isotherms a t 35' : curve A, unmodified wool; curve B, wool containing no amino groups; curve C, difference curve, representing contribution of amino groups to the wool-water isotherm.
tween this isotherm and the isotherm of unmodified wool is represented by curve C of Figure 3. This curve may be considered as the water vapor isotherm of the amino groups in keratin. The curve no longer has a pronounced sigmoidal shape and may represent an isotherm of the Langmuir type for monomolecular absorption. For a Langmuir isotherm there is a linear relation between the reciprocal of the amount absorbed and the reciprocal of the relative pressure. Curves A and C of Figure 3 are replotted in this way to give the corresponding curves A and C of Figure 4. (19) L.Valentine, Ann. Sci. TeztiEes Belges, 4, 206 (1956).
ROLEOF AMINOGROUPSIN WATERABSORPTION BY KERATIN
/-a
I
16
RECIP80CAL
I
I
24 RELATIVE PAESWRE
I
48
Figure 4. Reciprocal water content vs. reciprocal relative pressure for curves of Figure 3: curve A, unmodified wool; curve C, contribution of the amino groups.
The dotted line is an extrapolation of the linear portion of curve C. For curve C the relation is linear up to 25% relative humidity, but a t higher pressures there is a greater uptake than would be expected for a simple Langmuir isotherm. The Langmuir plot for unmodified wool (curve A of Figure 4) shows a very limited linear region. A Langmuir isotherm implies that there is a definite number of sites available for absorption and that each site is independent of its neighbors. Deviations from the Langmuir isotherm a t high water vapor pressures may occur for several reasons. For example, if swelling of the wool makes the amino groups accessible to more than one water molecule, the ratio of new sites becoming available to the number of existing unfilled “Langmuir” si1es may become appreciable. It is also possible that multimolecular absorption occurs a t high relative humidities. On the other hand, interaction between the absorbed water molecules may occur when a large proportion of the available sites becomes occupied. The linear portion of curve C, Figure 4, can be extrapolated to zero reciprocal relative pressure. From the intercept on the ordinate the saturation value can be calculated to be approximately 3.4% water content. This corresponds to the value obtained a t 70% relative humidity for the amino groups represented by curve C of Figure 3. The average number of water molecules associated with each aniino group a t various water vapor pressures can be calculated. At 25% relative humidity, where the amino group absorption deviates from the Langmuir isotherm, there are approximately 1.4 water molecules per amino group, while at 65% relative humidity a value of approximately 2.6 water molecules per amino group is obtained. This latter value is in excellent agreement
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with that arrived a t by Mellon, et U Z . , ~ for amino groups in casein. The amino group is capable of forming three hydrogen bonds,20 so a completely accessible group would probably associate with three water molecules. This saturation of amino groups occurs a t approximately 80% relative humidity (from curve C of Figure 3). Above 80% relative humidity the additional absorption probably represents attachment of water to water already on the amino groups. The extrapolation of the Langmuir plot (curve C, Figure 4) predicts a value of 2.7 water molecules for each amino group at saturation. Therefore, any interaction between the three possible water molecules has not prevented the available sites from being utilized; Le., the energy of interaction between sorbed water molecules is secondary in value to the energy of interaction between amino groups and water molecules. The high energy of bonding of 16.8 kcal./mole of water to an amino group would only be true for one water molecule. It is possible that a single water molecule could be attached through two, or even three, hydrogen bonds to an amino group at low humidities, resulting in a high energy of attachment. Xuclear magnetic resonance studies21 and measurement of activation energies of drying17 have shown that there is a smooth decrease in energy of binding of water to keratin with increase in water content, indicating that each increment of water reduces the average strength of attachment of water molecules already present in the wool. However, this will have no effect on the low humidity region of the isotherm. The attachment of water t o specific groups in wool a t humidities up to 80% relative humidity can be equated with these changes in energy of binding as follows. For amino groups, initial absorption from dryness would occur with high energy of binding; whereas, when two and finally three water molecules become associated wit.h each amino group as sorption proceeds, the average energy of binding of each water molecule would be expected to decrease. There would not be sharp changes in experimentally observed binding energies as absorption increased from 1 to 2 to 3 water molecules per amino group since, at any particular humidity, there would be certain proportions of amino groups having one, two, and three associations with water molecules, depending on the energy of attachment and the accessibility and steric environment of each particular amino group. (20) 0. L.Sponsler, J. D. Bath, and J. W. Ellis, J . Phvs. Chem., 44, 996 (1940). (21) G. W. West, A. R. Haly, and hl. Feughelman, Teztile Res. J., 31, 899 (1961).
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JAMESE. HUHEEY
3284
Thus, binding of water by amino groups constitutes a large percentage of the over-all sorption capacity of wool and is proportionately greater at low humidities. This result is in contrast to the deduction of BreuerZ2 that peptide groups are the primary water-binding sites. Other hydrophilic side chains and the peptide groups all contribute to water absorption23but not to a greater extent than the amino side chains, especially at low humidities. Previous worka has shown that “solution” or “condensation” absorption becomes a measurable component of the water content at 80% relative humidity; i.e., incoming sorbate molecules condense on water already present in the wool, so the present results may indicate that attachment of water to specific sorption sites is the main mechanism of sorption up to as high as 80% relative humidity, multimolecular absorption only occurring at higher pressures. It has been sho~n2~-26that volume swelling is less than volume sorption a t low water contents, and does not become proportional t,o volume sorption until 20% water con-
tent, ie., a t 80% relative humidity for unmodified wool. This reduced swelling is partly due to filling of voids and partly due to “electrostriction” of water molecules around charged groups in the protein. 2a It is significant that electrostriction of water by the amino groups would only occur up to the point where they become saturated with water molecules. Above 80% relative humidity, swelling is proportional to volume absorption, and it is in this region that electrostriction by the amino groups is no longer operative. 27p
(22) M. M. Breuer, J . Phys. Chem., 68, 2067 (1964). (23) J. D. Leeder and I. C. Watt, in preparation. (24) F. L. Warburton, J . TeztiEeInst. Trans., 38, T65 (1947). (25) J. L. Morrison and J. F. Hanlan, Nature, 179, 528 (1957). (26) J. H . Bradbury and J. D. Leeder, J . AppE. Polymer Sci., 7 , 545 (1963). (27) J. T. Edsall in ”The Proteins,” Vol. l B , H. Neurath and K. Bailey, Ed., Academic Press, Ino., New York, N. Y . , 1953, p. 565. (28) J. H. Bradbury, J . AppE. Polymer Sci., 7 , 557 (1963).
The Electronegativity of Groups
by James E. Huheeyl Division of Chemistry, Worcester Polytechnic Institute, Worcester, Massachueetta (Received February 16, 1966)
The electronegativities of 99 groups have been calculated by assuming variable electronegativity of the central atom in the group and equalization of electronegativity in all bonds. The resulting values are compared with those obtained by previous methods. It is suggested that one of the most important aspects of the electronegativity of groups is the relatively low values of the charge coefficients which have the effect of promoting charge transfer.
Electronegativity was originally defined2 as an invariant property of atoms. Recently, several workerss-’ have suggested that the electronegativity of an atom depends upon the environment of that atom in a molecule. For example, Walsha concluded that the electronegativity of carbon was dependent upon the hybridization of the atom. Sanderson4 suggested The Journal of Physieal Chemistry
that the electronegativity of an element depended upon its oxidation state, and Pritchard and Sumner5 in(1) Department of Chemistry, University of Maryland, College Park, Md. (2) L. Pauling and D. M. Yost, Proc. Natl. Acad. Sci. U. S., 14, 414 (1932); L. Pauling, “The Nature of the Chemical Bond,” 3rd Ed., Cornel1 University Press, Ithaca, N. Y . , 1960.
