Biochimica et Biophysica Acta, 322 (I973) 269-278
`© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
`
`BBA 36524
`
`A COMPARATIVE STUDY OF THE PHYSICOCHEMICAL PROPERTIES
`OF HUMAN KERATINIZED TISSUES
`
`HOWARD P. BADEN, LOWELL A. GOLDSMITH AND BARBARA FLEMING
`
`Department of, Dermatology, Harvard Medical School and Massachusetts General Hospital, Boston,
`Mass. (U.S.A.)
`
`(Received April 4th, 1973)
`
`SUMMARY
`
`Stratum corneum, hair and nail are all derived from ectodermal cells but show
`significant structural differences in their fully differentiated form. However, X-ray
`diffraction studies indicate that they all contain an a-fibrous protein with the same
`molecular dimensions. The greater tensile strength and stability to heating of hair
`and nail compared to stratum corneum can be explained by their higher content of
`half cystine. Studies of the isolated structural proteins indicate that the appendages
`contain a non-helical matrix component very rich in cystine while stratum corneum
`does not. Furthermore, stratum corneum has an a-fibrous protein with physico-
`chemical properties quite different from those of hair and nail, which are very similar
`to one another. Despite different morphological characteristics, it appears that hair
`and nail have differentiated along very similar lines. However, subtle differences in
`the relative proportion and composition of the structural proteins can be detected.
`A distinguishing feature of stratum corneum is its large content of lipids which make
`it an effective barrier to the diffusion of water.
`
`INTRODUCTION
`
`Keratinized tissues exhibit considerable heterogeneity in composition but they
`all contain structural proteins as major constituents12. In mammals these structural
`proteins are a mixture of a-fibrous and globular proteins in varying proportions~,4.
`Studies of wool proteins have demonstrated considerable species variation in physico-
`chemical properties, and in addition the fibrous and non-fibrous components them-
`selves are heterogeneous5. No such data is available, however, on human hair, stratum
`corneum and nail plate.
`A recent report on the harlequin fetuss, a genetic disorder of keratinization,
`has indicated that there can be an abnormality of the fibrous proteins of epidermis
`without a similar change in hair. This suggests that the formation of fibrous proteins
`in the epidermis and appendages may be under the control of separate genes. One
`might anticipate, therefore, that differences could be observed in the fibrous protein
`
`
`
`
`
`
`ARGENTUM EX1028
`
`Page 1
`
`

`

`of different keratinized tissues and perhaps in their non-fibrous components as well.
`Crounse7 compared the alkali-soluble proteins of hair, epidermis and nail and demon-
`strated striking similarities. The analyses were done on whole tissue, however, without
`prior separation of the different protein components. Development of newer tech-
`niques for tile identification of the various structural proteins of keratinized tissue
`has made it possible to compare more precisely the proteins of epidermis, nail and
`hair. The purpose of this report is to compare the structural protein of human
`stratum corneum, nail and hair and relate these to the differences and similarities in
`tile physical properties of these tissues.
`
`MATERIALS AND METHODS
`
`All tile chemicals used were of reagent grade except iodoacetic acid which was
`crystallized from anhydrous ether and light petroleum. Hair and nail clippings were
`obtained from normal individuals and washed with light petroleum before being used.
`Epidermis was separated from autopsy skin heated at 5o°C for 3o s and its under-
`surface was scraped with a scalpel to remove adherent epidermal cells. The remaining
`membrane consisted entirely of stratum corneum.
`
`Extraction procedures
`The various tissues were homogenized in 20 vol. of 0 M urea in o.I M Tris,
`pH 9.o, (Tris urea) and stirred at room temperature for 24 h. Following centrifu-
`gation the extraction was repeated a second time. The undissolved pellet was then
`extracted (io mg/ml) at 5° °C for 2 h under nitrogen in Tris-urea with o.I M mer-
`captoethanol for stratum corneum and 0.2 M mercaptoethanol for hair and nail. The
`suspension was centrifuged and an aliquot of the supernatant treated with iodoacetic
`acid to give the S-carboxymethyl derivatives. We have determined that these con-
`ditions give complete reduction and blockage of cystine residues. The alkylated and
`remaining untreated extracts were then dialyzed against distilled water and lyo-
`philized.
`
`X-ray diffraction
`X-ray diffraction analysis was done using nickle-filtered copper Ka radiation
`(2 = 1.54 A) at 4° kV at a specimen to film distance of 1.5o cm. Regenerated filaments
`for X-ray diffraction analysis were prepared by dissolving the protein in 8o % formic
`acid, picking up the solution on the tip of forceps and stretching while drying at
`room temperature.
`
`Stress-strain analysis
`Samples of hair were held between two sets of nylon clamps, one attached to a
`Stratham strain gauge (Stratham Lab., Los Angeles) and the other to a variable speed
`motor. The hair could be immersed in water or the whole apparatus placed in a
`chamber in which the humidity could be regulated. The specimen was stretched at
`constant rate and the tension recorded. Sonic velocity was measured by a dynamic
`modulus tester (L. M. Morgan Co., Cambridge, Mass.) as previously described9. The
`modulus of elasticity of nail was measured by deflection of the nail with different
`weights~°.
`
`Page 2
`
`

`

`PHYSICOCHEMISTRY OF KERATINIZED TISSUES
`
`271
`
`Water holding
`The water holding capacity was determined by suspending specimens from
`calibrated quartz springs in glass chambers in a constant temperature bath held at
`25 °C. The weight of the sample was determined by measuring the extension of the
`spring with a cathetometer. The humidity was controlled by placing anhydrous
`CaSO4 or saturated solutions of the various salts in the bottom of the chambersu.
`
`Water diffusion
`The diffusion of water was measured with aluminium chambers filled with
`water to which the specimen was fixed as a membrane. The chambers were stored
`with the specimen in contact with the water in desiccators at room temperature and
`weighed periodically to determine the rate of water loss.
`
`Amino acid analysis
`Samples for amino acid analysis were hydrolyzed in 6 M HC1 for 24 h under
`vacuum at IiO °C and run in duplicate on a Beckman 116 amino acid analyzer.
`
`Electrophoresis
`Disc electrophoresis was done at pH 8.3 using a 7% acrylamide gel with and
`without 6 M urea12.
`
`S content
`Total S content was determined gravimetrically following its oxidation to
`SO4~ and the addition of Ba (Belmont Analytical Lab,).
`
`RESULTS
`
`X-ray diffraction
`The results of X-ray diffraction analysis of human hair, nail and stratum
`corneum are shown in Table I. Although the same reflections are observed in hair
`and nail, the reflections in the former are much sharper indicating a higher degree of
`orientation. Unstretched stratum corneum shows only unoriented halos when the
`X-ray beam is perpendicular to the surface of the specimen but a partially oriented
`a pattern when it is parallel. After stretching the specimen the a pattern shows much
`sharper reflections. The orientation of filaments is parallel to the growth axis in hair
`
`TABLE I
`
`WIDE ANGLE X-RAY DIFFRACTION REFLECTIONS OF STRATUM CORNEUM, HAIR AND NAIL
`
`Very weak reflections were found at 4.15 ~ and/or 4.39 ~ (somewhat accentuated on the
`meridion} in some specimens of hair and nail and are likely due to c~ntarnination with scap1°.
`
`Equatorial Meridional
`reflection
`reflections
`A
`A
`
`Stratum corneum
`(oriented)
`Nail
`Hair
`
`9,8
`9.8
`9.8
`
`4.15, 5.14
`5. t 4
`5.14
`
`Page 3
`
`

`

`272
`
`H.P. BADEN t:l a[.
`
`and perpendicular in nail, while in epidermis there is only planar orientation unless
`the specimen is stretched. In epidermis an additional intense reflection can be seen
`at 4.15 ~ which is meridional in position with stretched tissue. Treatment of the tissue
`with polar organic solvents removes this reflection and it has been shown the material
`producing it has the characteristics of a lipidla.
`
`Water holding capacity
`The water holding capacity of stratum corneum, hair and nail at different
`relative humidities is shown in Fig. x. Much more water is held by stratum corneum
`than by hair or nail at high humidity. The data at low humidity is less reliable because
`of difficulty in reaching equilibrium conditions. Washing the tissues with a chloro-
`form-methanol mixture (3:x, v/v) followed by soaking in water markedly reduces
`the uptake of water by stratum corneum, but has no measurable effect on the other
`two tissues.
`
`!
`
`4°t
`
`60
`
`\
`
`0 20 40 60 80 I 0 0
`9~ RELATIVE HUMIDITY
`
`Fig. i. Water content of stratum corneum, hair and nail at different relative humidities. 0,
`stratum corneum; A, stratum corneum extracted with a chloroform-methanol mixture; ×, nail
`and hair which gave identical results.
`
`Water diffusion
`The flux of water across stratum obtained from skin of the anterior abdominal
`wall is in the range o.14 to 0.35 mgicm2 per h. Values almost ten times higher than
`this (2.0-3.0 mg!cm2 per h) are observed for nail plate. Since the thickness of stratum
`corneum is about I/IOO that of nail, the diffusion constant of water is several hundred
`times greater for nail compared to stratum corneum. No effect of chloroform-
`methanol (3 :z, v/v) extraction and soaking in water is observed for water flux in
`nail but the value for stratum corneum is increased almost Io-fold (z.9-3.2 mg/cm2
`per h) with this treatment.
`
`Response to heating in water
`The X-ray diffraction pattern of stratum corneum, nail and hair was determined
`before and after heating in water at 85 °C (ref. I4). No change is observed with hair
`and nail but with stratum corneum the pattern is lost and replaced by a cross fl one.
`Further heating of hair and nail produces no change until I3o °C when a poorly
`
`Page 4
`
`

`

`PHYSICOCHEMISTRY OF KERATINIZED TISSUES
`
`273
`
`oriented parallel fl pattern is noted in place of the normal a pattern. Isometric con-
`traction studies of stratum corneum show a marked increase in tension starting at
`about 80 °C but no contraction is observed with nail and hair even when the heating
`is continued to 95 °C.
`
`Modulus of elasticity
`The modulus of elasticity can be determined by several techniques. The
`measurement of the modulus of elasticity by sonic velocity techniques permits a
`direct comparison of all three tissues and the modulus of elasticity for stratum cor-
`neum, hair and nail are shown in Table II. The latter two have a much higher modulus
`
`TABLE I1
`
`YOUNG’S MODULUS FOR NAIL, HAIR AND STRATUM CORNEUM BY THE SONIC VELOCITY AND
`
`MECHANICAL TESTS
`
`Some oriention of abdominal stratum corneum occurs during drying of the specimens.
`
`Number off
`specimens
`
`Young modulus
`(dynesicm~ × zo-1° :~ S.D.)
`
`Sonic velocity
`technique
`
`Mechanical stretching
`
`50% R.H.
`
`zoo% R.H.
`
`70% R.H.
`
`Nail 12
`Hair
`13
`Stratum corneum
`Abdominal 2
`Sole
`2
`
`4.3 ± 0.4
`8.8 4- 0.6
`
`1,8 4- 0.5
`1.5 4 o.2
`
`2.6 -k 0.4
`2.3 ~- 0.3
`
`1.2 4- o.68
`0.47 4- o.15
`
`o,13 ~_ 0.07
`
`o,i9 4- o.04
`
`than the former. This same difference is also observed when mechanical methods of
`measurement are used and these relative differences are also apparent at different
`degrees of hydration of the tissue. Extraction of the tissue with a polar organic solvent
`(chloroform-methanol, 3 :i, v/v) does not alter the results indicating that the differ-
`ences are related to the structural proteins and their intrinsic organization.
`
`Amino acid analysis of whole tissue
`The amino acid composition of hair, stratum corneum and nail is shown in
`Table III. Although both hair and nail show a much higher content of half cystine
`than epidermis, the value for hair is higher than that for nail. Analyses of total S
`confirm these differences in half cystine content.
`
`Extraction of proteins
`The solubility of tissue proteins was studied in hair, nail and stratum corneum
`by first extracting the tissue with Tris-urea and then with the same buffer with the
`addition of o.I M mercaptoethanol. The results are shown in Table IV and indicate
`that in all three tissues the addition of a reducing agent markedly increases the yield
`of protein. The Tris-urea buffer did extract more protein from stratum corneum,
`however, and about half of this remained soluble when the extract was dialyzed
`against a neutral salt solution. Electrophoretic patterns of the urea-soluble proteins
`
`Page 5
`
`

`

`TABLE I[1
`
`AMINO ACID ANALYSIS OF HAIR, NAIL AND STRATUM CORNEUM
`
`The values given are the mcan of four different samples and arc expressed as residues per too
`residues. The S content is percent dry weight.
`
`A mino acid
`
`Hair
`
`Nail
`
`Stral~m
`
`COF~ebtYn
`
`Lysine
`Histidine
`Arginine
`Aspartic acid
`Threonine
`Serine
`Glutamic acid
`Proline
`Glycine
`Alanine
`Valine
`Methionine
`Isoleucine
`l+eucine
`T yrosine
`Phenylalaninc
`Halfcystine
`
`2.5
`0.9
`6.5
`5.4
`7-1)
`I2,2
`I2.2
`8.4
`5.8
`4.3
`5-5
`0.5
`2.3
`6, t
`2.2
`1.7
`~5.9
`o /
`¯ 4.5/o
`
`3. r
`~.o
`6.4
`7.o
`0. I
`iI.3
`I3.6
`
`4.2
`1.5
`3.8
`7.9
`3"O
`13.6
`I2.6
`
`5.9
`7.9
`5.5
`4.2
`0.7
`2.7
`8.3
`,3- z
`2.5
`1o.6
`~, o,
`3, - ..’o
`
`,3.o
`24.5
`4.4
`3.o
`t, [
`2.7
`6,9
`3.4
`3.2
`1.2
`¯ ~., o
`I t o/
`
`i ndicate that there are several components. X-ray diffraction patterns of the urea-
`s oluble proteins show no evidence of an a pattern, but a cross f! pattern is recognizable
`in a pH 2.65-soluble and pH 7.o-insoluble fraction from stratum corneum15.
`In the case of alt three tissues oriented filaments which give an a pattern by
`X-ray diffraction analysis can be prepared from the proteins solubilized by the buffer
`containing mercaptoethanol. The extracted proteins were converted to the S-carboxy-
`methyl derivatives to prevent the disulfide bonds from reforming. In order to solu-
`bilize the tyophilized S-carboxymethyl proteins with buffers in the pH range 7.o to
`9.o, urea at a concentration of 6 M has to be added. The nail and hair proteins remain
`in solution at concentrations up to at least 1% upon removal of the urea, but the
`stratum corneum proteins precipitate out of solution unless they are at a concen-
`tration below about o.1%. Disc electrophoresis of the S-carboxymethyl proteins
`
`TABLE IV
`
`AM[OUNT OF PROTEIN SOLUBILIZEI) BY DIFFERENT EXTRACTION PI~.OCEIIURPLS
`
`The tissue was treated sequentially with the two solvents. The data are expressed as perc~ J ! of
`initial dry weight of tissue.
`
`Tissue
`
`Percent yield -k S,D.
`
`6 ~FI urea with
`o.± M Tris (pH 9.o)
`
`6 3I urea with o.s ~’a/l
`Tris (pH 9.o) and o,s 3/1
`mercaptoethanol
`
`Stratum corneum
`Hair
`Nail
`
`25 -~ 5
`3 ~ 2
`I2 ± 4
`
`40±6
`40±3
`38~4
`
`Page 6
`
`

`

`PHYSICOCHEMISTRY OF KERATINIZED TISSUES
`
`275
`
`shows only a diffuse band at the origin of the running gel. Electrophoresis done in
`the presence of 6 M urea, however, gives distinct bands (Fig. 2). The electrophoretic
`patterns of hair and nail appear to be identical and this was confirmed by using
`IO% gels 15 cm long and run for twice the usual time. The most anodal component
`from hair and nail can be separated from the others by its solubility at pH 4.5 in
`o.5 M KC1. Filaments prepared from the insoluble proteins show an a pattern while
`those from the soluble ones show only unoriented halos. Disc electrophoretic patterns
`
`H N H : S
`
`i
`Fig. 2. Disc electrophoretic patterns of hair and nail S-carboxymethyl proteins in 6 M urea. H,
`hair; N, nail.
`
`Fig. 3. Disc electrophoretic patterns of hair and stratum corneum S-carboxymethyl proteins in
`6 M urea. H, hair; S, stratum corneum.
`
`of a protein from stratum corneum (Fig. 3) in 6 M urea show a completely different
`pattern from those of nail and hair. When solutions of the protein are adjusted to
`pH 4.5 with the addition of 0.5 M KC1, only trace amounts remain soluble.
`Amino acid analysis of the ffactionated nail and hair proteins is shown in
`Table V and indicates the striking difference in composition especially with regard
`to half cystine between the pH 4.5-soluble and -insoluble proteins. The same fractions
`from hair and nail on the other hand show a close similarity. Amino acid analysis of
`the pH 4.5-insoluble proteins from stratum corneum, however, is much different than
`either fraction of hair and nail.
`
`Page 7
`
`

`

`276
`
`TABLE V
`
`n.P. BAI)E,N gl al.
`
`AMINO ACID ANALYSIS OF HAIR, NAIL AND STRATUM CORNEUM S-CARBOKVMI~THYL PROTEINS
`
`The a proteins are insoluble at pH 4,5 in the presence of 0.5 M KC1 while the matrix ones are
`soluble. The values are expressed as residues per Ioo residues.
`
`.,t mino acid
`
`Hair
`
`Nail
`
`Stratum corneum
`
`3lalrix
`
`~
`
`Matrix
`
`r~
`
`a
`
`2.9
`0.6
`7.0
`8,4
`5-9
`lO.5
`17,1
`5,6
`5.3
`6.0
`
`5-3
`0.6
`3.o
`9.8
`2.8
`2, I
`
`i.o
`o.8
`0.3
`3.2
`9.9
`13-1
`lo. ~
`lO,9
`3-9
`2.3
`4.8
`0.3
`1.8
`3,7
`
`I .(I
`
`1.3
`
`2.8
`o. 7
`7, i
`io.4
`3.9
`9.4
`14,8
`
`5,3
`8.8
`8.3
`3.9
`trace
`2.9
`lO.7
`
`3.0
`
`2,5
`
`0,7
`i. i
`5.o
`3.7
`lO.4
`I3.O
`8.7
`12,8
`6.9
`2.5
`4.3
`I.i
`1.9
`4,6
`
`1.0
`
`1.4
`
`7-1 25.0
`
`5.5 20-3
`
`Lysine
`Histidine
`Arginine
`Aspartic acid
`Threonine
`Serine
`Glutamic acid
`Proline
`Glycine
`Alanine
`Valine
`Methionine
`Isoleucine
`Leucine
`Tyrosine
`Phenylalanine
`S-carboxyrnethyl
`eysteine
`
`DISCUSSION
`
`4,3
`i.o
`4.8
`9.5
`3.9
`II.7
`13.7
`2,3
`18.0
`5.6
`3,7
`1.5
`3.2
`8.5
`3.2
`3.4
`
`I.O
`
`These studies indicate that there is a striking similarity in the physical and
`chemical properties of hair and nail, although there are some clear differences. Both
`show a similar modulus of elasticity by the sonic velocity as well as mechanical
`methods. The higher values obtained from hair using the sonic modulus reflects the
`higher degree of filament orientation and this is also shown by the sharper reflections
`observed in the X-ray diffraction patterns. Although in human nails the filaments
`are aligned perpendicular to the growth axis, the reverse of hair, this is not a general
`property of nails but appears to be related to their shapel°.
`Hair and nail are also quite similar in their capacity to absorb water at varying
`relative humidities. Although the diffusion of water through hair was not measured,
`data on the rate of hydration by measurement of swelling suggest that it must be
`relatively rapid and similar to nail. The lack of an effect of extraction with polar
`organic solvents and water is also similar for both tissues.
`The stability of the a helix to heating in water is also similar and no change
`in the a pattern is observed until the tissues are placed at 13o °C (refs IO and 16).
`This stability appears to be related in part to the organization and composition of
`the structural proteins. The configuration which appears after heating is a partially
`oriented parallel fl and the orientation, in the case of hair, can be improved by gentle
`stretching.
`The differences observed between the total S and half cystine content of hair
`and nail can be explained in part by variation in the amount of matrix component.
`The relative amount of protein precipitated at pH 4-5 with o.5 M KC1 is greater for
`
`Page 8
`
`

`

`PHYSICOCFIEMISTRY OF KERATINIZED TISSUES
`
`277
`
`hair than nail. It is also likely that some difference in the matrix component exists
`since the half cystine contents appear to be different.
`Stratum corneum shows both quantitative and qualitative differences from
`hair with respect to many of its physicochemical properties. The a filaments are
`present both in the appendageal tissues and stratum corneum, but in the latter these
`structures lie parallel to the surface, with no specific directional orientation unless
`the tissue is stretched. The organization of the filaments and their relationship to
`matrix components is also quite different and this is reflected in a number of para-
`meters. The modulus of elasticity as measured both by the sonic and mechanical
`methods is much lower than for hair or nail. Isometric contractions of epidermis with
`melting of the a helix and conversion to a cross/~ structure occurs at about 80 °C,
`while hair and nail show change in configuration at 13o °C and form a partially
`oriented parallel ft. These differences are certainly related in part to the organization
`of the filaments and the cross-links between them and matrix proteins, but differences
`in the filamentous structures themselves may be a factor, The a proteins isolated
`from stratum corneum behave as macromolecular aggregates and can not be easily
`separated into a number of distinct components as can hair and nail. The proteins
`from stratum corneum show a considerable difference in amino acid composition from
`those of the appendages and this is particularly true with respect to half cystine.
`Considering the much lower values of half cystine in stratum corneum proteins and
`the care taken to completely reduce and block the bonds, it is difficult to explain the
`tendency toward aggregation on residual cystine bonds. The presence of other cross-
`links is suggested by the finding of blocked e-lysine groups in the fibrous proteins of
`stratum corneum and these may explain some of the differences in physical proper-
`ties17.
`
`The structural proteins of stratum corneum also differ from those of the
`appendages with regard to the matrix components. No distinct proteins rich in
`cystine have been identified in the stratum corneum. The insolubility of the structural
`proteins, however, is dependent on disulfide cross-links. It has been shown earlier
`that intermolecular cystine cross-links are not present in the prekeratin filaments of
`the viable epidermis but are formed during the latter stages of keratinization and are
`responsible for the change in solubility of the filamentsis. That the number and
`perhaps character of the cystine bonds differs in stratum corneum compared to the
`appendages may be reflected in their varying response to temperature both with
`respect to the temperature of melting of the helix and change in molecular con-
`figuration. These findings indicate that there are significant differences in the inter-
`action of the varying structural proteins in these different keratinizing tissues, al-
`though the same cross-link appears to be important in both.
`One of the most striking differences between stratum corneum and the ap-
`pendages is in the absorption and diffusion of water. At high relative humidities
`stratum corneum absorbs several-fold more water than the other two tissues. This
`appears to be related to the presence of water absorbing materials which can be
`removed by the extraction of the tissue with polar organic solvents and water.
`Following such treatment water absorption is similar to hair and nail, which are
`unaffected by the extraction procedures. The lower diffusion constant of water in
`stratum corneum compared to nail (and presumably hair) is also increased by the
`extraction procedure. It has been suggested that these properties of stratum are
`
`Page 9
`
`

`

`278
`
`H.P. BADEN et al.
`
`dependent on the presence of a particular lipid which is responsible for the barrier
`characteristic. However, attempts to identify a specific lipid by chromatographic
`techniques were not successfuPu. Baden and Goldsmith13 were able to demonstrate
`the presence of a lipid component by X-ray diffraction techniques which appeared
`to have a distinct structural relationship to the filaments. From their observations
`it is possible to develop a bimolecular membrane modei similar to what has been
`described for other tissues. Recently Scheuplein and Morgan2° have offered an alterna-
`tive explaination. They have suggested that diffusion of water is limited by bound
`water and the role of lipid is to form a compIex with the tissue proteins and other
`water absorbing materials and thus maintain the bound water. Although the exact
`mechanism underlying the interaction of water with stratum corneum is not entirely
`dear, it must be different than that of hair and nail. This appears to be directly
`related to the important function of the stratum corneum as a diffusion barrier.
`These results indicate the extent of diversity which can be observed in kera-
`tinized tissues. Although derived from a common cell type, they can exhibit funda-
`mental differences in their fully differentiated form. This in part may explain how
`genetic disorders of keratinizing tissues can involve one type while the remainder
`appear to be uninvolved. Studies are in progress using the techniques described to
`study certain disorders of keratinizing tissues.
`
`ACKNOWLEDGEMENT
`
`This work was supported by N.I.H. grant No. AM o6838.
`
`REFERENCES
`
`I Crewther, k¥. G., Fraser, R. D. B., Lennok, F. G. and Londley, H. (1965) Adv. Prot. Chem. 20,
`191-346
`z Mercer, E. H. (I96I) Keratin and Keratinization, pp. 1 5I, Pergamon, New York
`3 Corfield, M. C. and Fletcher, J. C. (I968) ,~ymposium on Fibrous Proteins, pp. 299-313, Plenum,
`New York
`4 Thompson, E. O. P. and O’Donnell, I. J. (1965) Aust. J. Biol. Sci. t8, i2o7--i225
`5 Joubert, F. J., De Jager, p. J. and Swart, L. S. (1968) S~,mposium on Fibrous Proteins, pp.
`343-352, Plenum, New York
`6 Craig, J. M., Goldsmith, L. A. and Baden, H. P. (197o) Pediatrics 40, 437-44°
`7 Crounse, R. G. (~968) Biology of Skin and Hair Growth, pp. 3o7-312, Elsevier, New York
`8 Hirs, C. W. H. (1967) Methods in Enzymology, pp. I99-2o3, Academic Press, New York
`9 Goldsmith, L. A. and Baden, H. P. (I97o) J. Invest. Dermatol. 55, 256 259
`IO Baden, H. P. (I97o) J. Invest. Dermatol. 55, II5-122
`~i Rockland, 1,. B. (196o) Ancd. Chem. 32, 1375 1376
`12 Davis, B. J. (~964) Ann. N.Y. Acad. Sat. I11, 4o4-436
`~3 Baden, H. P. and Goldsmith, L. A. (I97o) Nature 225, Io52-IO53
`14 Gifford, A. M. and Baden, H. P. (197o) J. Invest. DermatoI. 54, 298--3o3
`15 Baden, H. P., Goldsmith, L. A. and Boner, L. C. (1973) J. Invest. DermaloL, in the press
`16 Feughelman, M. and Mitchell, T. "W. (1968) Biopolymers 6, 1515 1518
`17 Baden, H. P. and Goldsmith, L. A. (I972) Clin. Res. 2o, 416
`18 Baden, H. P. and Goldsmith, L. A. (1971) Acla Dermato-Venereol. 5I, 312 316
`19 Sweeney, T. M. and Downing, D. T. (i97o) J. Invest. Dermatol. 55, I39 144
`2o Scheuplein, R. J. and Morgan, L. J. (I967) Nature 2I4, 456 458
`
`Page 10
`
`