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On the keratin fibrils of the skin
S. ULTRASTRUCTURERESEARCH3, 51-57 (1959)
51
On the Keratin Fibrils of the Skin An X-ray Small Angle Scattering Study of the Horny Layer G. SWANBECK
Department of Medical Physics, Karolinska Instituter, Stockholm Received March 25, 1959 The horny layer of the skin has been investigated by means of X-ray diffraction. The fibrils of the horny layer are found to be independently scattering and are therefore assumed to be irregularly arranged. Slope- and peak-analysis of the oriented scatter gives a fbril diameter of 260 X. The aggregation of seven tonofibrils into the 260 A fibril of the horny layer is considered to be the main step in the keratinization process of the epidermis. Little is known about the ultrastructural organization of the fibrous component of the human horny layer. Some works are published on the ultrastructure of the germinative epithelium of epidermis investigated by means of electron microscopy (2, 23, 28, 32, 33). In these works tonofibrils of a thickness of 100 ~ are described. Rudall has published a very comprehensive work on the protein of the cow nose epidermis, f r o m which he has extracted a fibrous s-keratin called epidermin and a non-fibrous component giving a disoriented fl-pattern (31). The same author has also published an X-ray diffraction study of the horny layer of different animals and shows that fi-keratin can be found in the skin of birds and reptiles (30). The presence of ~keratin in epidermis of man was discovered already by Derksen and Heringa in 1936, and Derksen et al. in 1937 (8, 9). Giroud and Champetier published in 1936 an X-ray diffraction study involving epidermis (12). The terms ~-keratin and fl-keratin were introduced by Astbury et al. (cf. 1). Pauling and Corey (24) have proposed a now generally accepted model (the ~-helix) for the peptide chain in c~-keratin and some other proteins. The c~-keratin has been studied mainly in wool, hair and porcupine quill tips. MacArthur (16) and Bear (3, 4) have evaluated the X-ray diffraction long spacings of these keratin derivatives. Some comprehensive work has been made with electron microscopy and X-ray diffraction on wool and hair by Birbeck and Mercer (5-7) and Fraser et al. (10, 11). They have found regularly spaced fibrils of a thickness of 70 ~ and an osmiophilic interfibrillar substance.
52
G. SWANBECK
In spite of the great differences between soft and hard keratin, many dermatologists believe them to be different stages in a common keratinization process. Rothman (29) says in his valuable book: "In view of modern knowledge, it is obvious that keratinization is a continuous progressive process and that it is a matter of the degree of keratinization which determines how the end-products will behave. Therefore, it is rahter hopeless to establish categories." This work is intended to give information on the organization of the fibrous protein of the horny layer, and will be a base for further studies on the keratin of some desquamating dermatoses. The orientation of the fibrils of the horny layer has been investigated by polarization microscopy by Montagna (22), Matoltsy (17), Matoltsy and Odland (19, 20) and Matoltsy and Balsamo (18). Montagna states that the fibrils are oriented parallel to the surface of the skin while Matoltsy et al. have made a more thorough investigation in which, however, they seem to have misinterpreted the polarization microscopical pictures.
MICROSCOPY The following is a summary of what can be learnt from microscopical studies of the horny layer. The horny layer of the skin consists of flattened hornified cells. These cells are found to be 0.5-0.7 micron thick when cut at right angle to the plane of the cell and measured at ultrathin sections with the phase contrast microscope (34). The keratin is wholly intracellular. As the horny cells are 20-50 micron in diameter and less than one micron thick, the fibrils can be regarded as oriented only in one plane parallel to the plane of the cells. If the cells are not stretched, the fibrils are believed to be oriented at random in the plane of the cells. In such a case the cell is isotropic if viewed at right angle to the plane of the cell, but anisotropic i f viewed along the plane of the cell, and the birefringence is in this case 63.7 % of the value of an ideally oriented fibre consisting of the same material. The dry mass of stratum corneum is about 50 % by volume (21, 34) and the fibrous protein 65 % of the dry mass (17). The birefringence of the horny layer will then be about 20 % of the birefringence of an isolated fibril free from water. Measurements of birefringence of skin sections cut at right angle to the surface of the skin have given values of approx. 0.002-0.003 which is a fourth of the value for the consolidated hair cortex, where fibres are closely packed and well oriented. A section cut parallel to the surface of the skin will show birefringence in the vicinity of the sulci cutis, as the hornified cells will make an angle to the plane of the section thus giving a false impression of a fibril orientation along the epidermal furrows.
KERATIN FIBRILS OF SKIN
53
FI6.1. Low angle diffraction pattern showing a continuous elongated scatter. Two diffuse maxima indicated by arrows, x 6.
X-RAY DIFFRACTION
Material and methods For the quantitative measurements a slit collimated Guinier camera giving monochromatic CrK~ radiation was used. The width of the beam when striking the plane of the film was 0.1 ram. The estimation of the orientation and position of the maxima were made with a pinhole collimated, evacuated low-angle camera capable of resolving 400 A. In this camera Nifiltered Cu radiation was used. The specimens were taken from the foot sole of three adult persons. The preliminary wide-angle X-ray diffractograms show a completely unoriented a-pattern when the incident beam strikes the horny layer perpendicular to the surface of the skin, and a partly oriented c~-pattern when the incident beam strikes the horny layer parallel to the surface of the skin. This is in agreement with the polarization microscopical findings. The low-angle pin-hole collimated diffraction (incident beam parallel to the skin surface) showed a continuous scatter followed by two to three diffuse maxima having the same degree of orientation as the wide-angle diffractograms showed (Fig. 1). The observed continuous scatter indicates that the fibrillar organization must be very irregular. Either the fibrils are irregularly spaced and of uniform diameter or they are of different diameters. The weak diffuse maxima observed indicates that the fibril diameter is very constant, however. The maxima can either be Bragg reflections caused by aggregation of only a few of the fibrils or secondary maxima of the square of the shape function for the fibrils. As there is a marked orientation of the fibrils, no serious error will be introduced if the relative intensities are measured from a slit collimated camera.
Mathematical treatment of the independent scattering perpendicular to the axis of fibres with circular cross section. The scattered intensity I(hR) is proportional to the square of the structure factor F(hR). The scattered intensity will thus be
54
G. SWANBECK
I(hR)=k.FZ(hR)=k.
[2J-~(Rh~R)]2
(1)
where Jt is a Bessel function of the first order, k a constant, R the radius of the cross section of the fibril, and h = 2 re e/2, where s is the scattering angle. F 2 (hR) has secondary maxima for hR = 5.14, 8.46, 11.62 etc. Corresponding maxima of the scattering function for spheres have been used by Yudowitch for the estimation of the diameter of latex particles (35). For small values of hR the scattering function (1) can be approximated by R~h2
I(hR)=k.e
4
(2)
which is in analogy with Guinier's approximation for spherical particles (13, 14). A similar approximation has been derived by Porod (26, 27) for long cylinders with circular cross section oriented at random. The function f(e ~) = log k" e -~h"/4 will be linear and therefore the function g(e ~) = log I will be linear for small values of hR. From the slope of this curve for small values of hR the value ,of R can be calculated R = 0,483 2 ~/p
(3)
where 2 is the wavelength, and - p the slope of the curve. By derivating the function =
•
it can be tested how correct the approximationf(e 2) = log
k" e -~h014 is for different values of hR. These calculations have shown that when measuring the slope at hR = 1 the function f ( e 2) will give 1.5 % smaller value than g (e~) and at h R = 2 , f ( e ~) will give a 10.8 % smaller value than g (e~). In this work the measurements have been made just above hR = 1. This reasoning is applicable only when the fibrils are scattering independently. This is so in the case of dilute systems but seldom in densely packed systems. For densely packed systems interparticle interferences often modify the scattering curve. Porod (27), and Kratky and Porod (15) emphasize one exception from this rule. This is when fibrils are not lying strictly parallel but are running in different directions. The horny layer of the skin is precisely the most ideal densely packed system in which a good orientation of the diffractograms is obtained but at the same time the fibrils do not run parallel and conditions for independent scattering are obtained. The intensity of the continuous scatter near the beam-stop is about 100 times higher than the intensity of the first secondary maximum. The second maximum is weaker than the first and the third weaker than the second. When log I(h) is plotted against e ~, the curve has a linear portion near the beam-stop (Fig. 2). For this part of the curve - p equals 15000, which gives a fibril diameter of 270 ~. The two fol-
KERATIN FIBRILS OF SKIN
55
O
Log]i
~
, e
o
-I Fla. 2. The logarithm of the intensity along the long axis of the continuous scatter plotted versus square of the scattering angle.
lowing diffuse maxima give, with peak-analysis, a fibril diameter of 250 • and 260 ~ respectively. The discrepancy between these values is small and presumably due to the difficulty in determining the exact position of the maximum point. The fact that the curve log I(h) against e2 has a straight portion near the direct beam is a criterion of the fibrils being irregularly packed and of uniform size. The appearance of the secondary maxima is a criterion of the fibril thickness being highly uniform and of the cross section of the fibrils being circular. As two different methods, slope analysis and peak analysis, made with two different diffraction equipments (the slope analysis with a slit collimated camera with CrKc~ radiation and the peak analysis with a pin-hole collimated camera with CuKc~ radiation) give values that are in close agreement, a fibril diameter of 260 A must be very probable. DISCUSSION The correlation of these results with the body of information already known about the epidermis can only be made on a speculative basis. As mentioned above the electron microscopists have found a diameter of 100 A on the tonofibrils of epidermis. However, the diameter found in this investigation is about 3 times larger for the fibrils of the horny layer. It is therefore probable that several aggregating tonofibrils build up the 260 ~ fibril of the horny layer. One of the main processes in the keratinization of epidermis is perhaps the aggregation of the tonofibrils into the 260 A fibrils. The fact that the 260 A fibrils are scattering independently means that these fibrils are not regularly packed but probably freely movable with respect to each other. A regular packing of the fibrils would possibly lead to a hard and brittle skin as in some dermatoses. An investigation of the molecular packing in some dermatoses is now in progress. It is evident that the molecular arrangement is quite different in the
56
G. SWANBECK
horny layer of the skin and in the hair-cortex. This difference is already present in the germinative epithelium where the epidermis has 100 A tonofibrils and the hair corresponding fibrils of 70 A, which pack together in large bundles of some thousand A. It is very difficult if not impossible to investigate the packing of the peptide chains or the c~-helices in the tonofibrils, because of the irregular arrangement of the fibrils. If the model proposed by Pauling and Corey (25) for the arrangement of the ~helices in s-keratin with six helices twisted around one helix as a seven strand rope is true, it can be assumed that these ropes or protofibrils build up seven strand cables which is the tonofibrils and that the tonofibrils also build up a seven strand cable making a 260 A fibril in the horny layer.
ACKNOWLEDGEMENTS We are grateful for financial support from the research grant No. AF61 (052)-21 from the European Office of Air Research and Development Command, U.S. Air Force, and grant D700, National Institutes of Health, Bethesda, Md., U.S.A.
REFERENCES
l. ASTBURY,W. T. and STREET,A., Phil. Trans. Roy. Soc. London Ser. A 230, 75 (1931). 2. BArm, G., ft'rztl. Forsch. 10, 255 (1956). 3. BEAR, R. S., J. Am. Chem. Soc. 65, 1784 (1943). 4. - ibid. 66, 2043 (1944). 5. BIRBECK, M. S. C., J. Biophys. Biochem. CytoI. 3, 203 (1957). 6. - ibid. 3, 215 (1937). 7. - ibid. 3, 223 (1957). 8. DERKSEN,J. C. and HERINGA,G. C., Polska Gaz. Lekarska 15, 592 (1936). 9. DERKSEN,J. C., HERINGA,G. C. and WEIDINGER,A., Acta Neerl. Morphol. 1, 31 (1937). 10. FRASER, R. D. B. and MACRAE, T. P., Biochim. et Biophys. Acta 29, 229 (1958). 11. FRASER,R. D. B., MACRAE, T. P. and ROGERS, G. E., Nature 183, 592 (1959). 12. GIROU~),A. and CHAMPETIER,G., Bull. soe. chim. biol. 18, 656 (1936). 13. GUINIER,A., Ann. phys. 12, 161 (1939). 14. GUINIER,A., FOtmNET, G., WALKER, C. B. and YUDOW~TC~, K. L., Small Angle Scattering of X-rays. John Wiley & Sons, New York, and Chapman & Hall, London, 1955. 15. KRAT~CY,O. and POROD, G., Z. physik. Chem. N.F. 7, 236 (1956). 16. MAcARTHUR, I., Nature 152, 38 (1943). 17. MATOLTSY,A. G., in MONTAGNA,W. and ELLIS, R. A. (Eds.), The Biology of Hair Growth, p. 135. Academic Press, New York, 1958. 18. MATOLTSY,A. G. and BALSAMO,C. A., J. Biophys. Biochem. CytoL 1, 339 (1955). 19. MATOLTSY,A. G. and ODLAND, G. :F., J. Biophys. Biochem. Cytol. 1, 191 (1955). 20. - J. Invest. DermatoL 26, ]21 (1956). 21. MOBERGER,G. and ENGSTR/3M,A., J. Invest. Dermatol. 22, 477 (1954).
KERATIN FIBRILS OF SKIN
57
22. MONTAGNA,W., The Structure and Function of Skin. Academic Press, New York, 1956. 23. ODLAND, G. F., J. Biophys. Biochem. Cytol. 4, 529 (1958). 24. PAULING, L. and COREY, R. B., Proe. Natl. Acad. Sci. U.S. 37, 205 (1951). 25. - Nature 171, 59 (1953). 26. POROD, G., Acta Phys. Austriaca 2, 255 (1948). 27. - Kolloid-Z. 124, 83 (1951). 28. PORTER, K. R., Anat. Record 118, 433 (1954). 29. ROTHMAN, S., Physiology and Biochemistry of the Skin. University of Chicago Press, Chicago, 1954. 30. RUDALL, K. M., Bioehim. et Biophys. Acta 1, 549 (1947). 31. - Advances in Protein Chem. 7, 253 (1952). 32. SEL~Y, C. C., J. Biophys. Biochem. Cytol. 1, 429 (1955). 33. - J. Soc. Cosmetic Chemists 7, 584 (1956). 34. SWANBECK,G., unpublished. 35. YUDOW~TCH,K. L., J. Appl. Phys. 20, 174 (1949).
51
On the Keratin Fibrils of the Skin An X-ray Small Angle Scattering Study of the Horny Layer G. SWANBECK
Department of Medical Physics, Karolinska Instituter, Stockholm Received March 25, 1959 The horny layer of the skin has been investigated by means of X-ray diffraction. The fibrils of the horny layer are found to be independently scattering and are therefore assumed to be irregularly arranged. Slope- and peak-analysis of the oriented scatter gives a fbril diameter of 260 X. The aggregation of seven tonofibrils into the 260 A fibril of the horny layer is considered to be the main step in the keratinization process of the epidermis. Little is known about the ultrastructural organization of the fibrous component of the human horny layer. Some works are published on the ultrastructure of the germinative epithelium of epidermis investigated by means of electron microscopy (2, 23, 28, 32, 33). In these works tonofibrils of a thickness of 100 ~ are described. Rudall has published a very comprehensive work on the protein of the cow nose epidermis, f r o m which he has extracted a fibrous s-keratin called epidermin and a non-fibrous component giving a disoriented fl-pattern (31). The same author has also published an X-ray diffraction study of the horny layer of different animals and shows that fi-keratin can be found in the skin of birds and reptiles (30). The presence of ~keratin in epidermis of man was discovered already by Derksen and Heringa in 1936, and Derksen et al. in 1937 (8, 9). Giroud and Champetier published in 1936 an X-ray diffraction study involving epidermis (12). The terms ~-keratin and fl-keratin were introduced by Astbury et al. (cf. 1). Pauling and Corey (24) have proposed a now generally accepted model (the ~-helix) for the peptide chain in c~-keratin and some other proteins. The c~-keratin has been studied mainly in wool, hair and porcupine quill tips. MacArthur (16) and Bear (3, 4) have evaluated the X-ray diffraction long spacings of these keratin derivatives. Some comprehensive work has been made with electron microscopy and X-ray diffraction on wool and hair by Birbeck and Mercer (5-7) and Fraser et al. (10, 11). They have found regularly spaced fibrils of a thickness of 70 ~ and an osmiophilic interfibrillar substance.
52
G. SWANBECK
In spite of the great differences between soft and hard keratin, many dermatologists believe them to be different stages in a common keratinization process. Rothman (29) says in his valuable book: "In view of modern knowledge, it is obvious that keratinization is a continuous progressive process and that it is a matter of the degree of keratinization which determines how the end-products will behave. Therefore, it is rahter hopeless to establish categories." This work is intended to give information on the organization of the fibrous protein of the horny layer, and will be a base for further studies on the keratin of some desquamating dermatoses. The orientation of the fibrils of the horny layer has been investigated by polarization microscopy by Montagna (22), Matoltsy (17), Matoltsy and Odland (19, 20) and Matoltsy and Balsamo (18). Montagna states that the fibrils are oriented parallel to the surface of the skin while Matoltsy et al. have made a more thorough investigation in which, however, they seem to have misinterpreted the polarization microscopical pictures.
MICROSCOPY The following is a summary of what can be learnt from microscopical studies of the horny layer. The horny layer of the skin consists of flattened hornified cells. These cells are found to be 0.5-0.7 micron thick when cut at right angle to the plane of the cell and measured at ultrathin sections with the phase contrast microscope (34). The keratin is wholly intracellular. As the horny cells are 20-50 micron in diameter and less than one micron thick, the fibrils can be regarded as oriented only in one plane parallel to the plane of the cells. If the cells are not stretched, the fibrils are believed to be oriented at random in the plane of the cells. In such a case the cell is isotropic if viewed at right angle to the plane of the cell, but anisotropic i f viewed along the plane of the cell, and the birefringence is in this case 63.7 % of the value of an ideally oriented fibre consisting of the same material. The dry mass of stratum corneum is about 50 % by volume (21, 34) and the fibrous protein 65 % of the dry mass (17). The birefringence of the horny layer will then be about 20 % of the birefringence of an isolated fibril free from water. Measurements of birefringence of skin sections cut at right angle to the surface of the skin have given values of approx. 0.002-0.003 which is a fourth of the value for the consolidated hair cortex, where fibres are closely packed and well oriented. A section cut parallel to the surface of the skin will show birefringence in the vicinity of the sulci cutis, as the hornified cells will make an angle to the plane of the section thus giving a false impression of a fibril orientation along the epidermal furrows.
KERATIN FIBRILS OF SKIN
53
FI6.1. Low angle diffraction pattern showing a continuous elongated scatter. Two diffuse maxima indicated by arrows, x 6.
X-RAY DIFFRACTION
Material and methods For the quantitative measurements a slit collimated Guinier camera giving monochromatic CrK~ radiation was used. The width of the beam when striking the plane of the film was 0.1 ram. The estimation of the orientation and position of the maxima were made with a pinhole collimated, evacuated low-angle camera capable of resolving 400 A. In this camera Nifiltered Cu radiation was used. The specimens were taken from the foot sole of three adult persons. The preliminary wide-angle X-ray diffractograms show a completely unoriented a-pattern when the incident beam strikes the horny layer perpendicular to the surface of the skin, and a partly oriented c~-pattern when the incident beam strikes the horny layer parallel to the surface of the skin. This is in agreement with the polarization microscopical findings. The low-angle pin-hole collimated diffraction (incident beam parallel to the skin surface) showed a continuous scatter followed by two to three diffuse maxima having the same degree of orientation as the wide-angle diffractograms showed (Fig. 1). The observed continuous scatter indicates that the fibrillar organization must be very irregular. Either the fibrils are irregularly spaced and of uniform diameter or they are of different diameters. The weak diffuse maxima observed indicates that the fibril diameter is very constant, however. The maxima can either be Bragg reflections caused by aggregation of only a few of the fibrils or secondary maxima of the square of the shape function for the fibrils. As there is a marked orientation of the fibrils, no serious error will be introduced if the relative intensities are measured from a slit collimated camera.
Mathematical treatment of the independent scattering perpendicular to the axis of fibres with circular cross section. The scattered intensity I(hR) is proportional to the square of the structure factor F(hR). The scattered intensity will thus be
54
G. SWANBECK
I(hR)=k.FZ(hR)=k.
[2J-~(Rh~R)]2
(1)
where Jt is a Bessel function of the first order, k a constant, R the radius of the cross section of the fibril, and h = 2 re e/2, where s is the scattering angle. F 2 (hR) has secondary maxima for hR = 5.14, 8.46, 11.62 etc. Corresponding maxima of the scattering function for spheres have been used by Yudowitch for the estimation of the diameter of latex particles (35). For small values of hR the scattering function (1) can be approximated by R~h2
I(hR)=k.e
4
(2)
which is in analogy with Guinier's approximation for spherical particles (13, 14). A similar approximation has been derived by Porod (26, 27) for long cylinders with circular cross section oriented at random. The function f(e ~) = log k" e -~h"/4 will be linear and therefore the function g(e ~) = log I will be linear for small values of hR. From the slope of this curve for small values of hR the value ,of R can be calculated R = 0,483 2 ~/p
(3)
where 2 is the wavelength, and - p the slope of the curve. By derivating the function =
•
it can be tested how correct the approximationf(e 2) = log
k" e -~h014 is for different values of hR. These calculations have shown that when measuring the slope at hR = 1 the function f ( e 2) will give 1.5 % smaller value than g (e~) and at h R = 2 , f ( e ~) will give a 10.8 % smaller value than g (e~). In this work the measurements have been made just above hR = 1. This reasoning is applicable only when the fibrils are scattering independently. This is so in the case of dilute systems but seldom in densely packed systems. For densely packed systems interparticle interferences often modify the scattering curve. Porod (27), and Kratky and Porod (15) emphasize one exception from this rule. This is when fibrils are not lying strictly parallel but are running in different directions. The horny layer of the skin is precisely the most ideal densely packed system in which a good orientation of the diffractograms is obtained but at the same time the fibrils do not run parallel and conditions for independent scattering are obtained. The intensity of the continuous scatter near the beam-stop is about 100 times higher than the intensity of the first secondary maximum. The second maximum is weaker than the first and the third weaker than the second. When log I(h) is plotted against e ~, the curve has a linear portion near the beam-stop (Fig. 2). For this part of the curve - p equals 15000, which gives a fibril diameter of 270 ~. The two fol-
KERATIN FIBRILS OF SKIN
55
O
Log]i
~
, e
o
-I Fla. 2. The logarithm of the intensity along the long axis of the continuous scatter plotted versus square of the scattering angle.
lowing diffuse maxima give, with peak-analysis, a fibril diameter of 250 • and 260 ~ respectively. The discrepancy between these values is small and presumably due to the difficulty in determining the exact position of the maximum point. The fact that the curve log I(h) against e2 has a straight portion near the direct beam is a criterion of the fibrils being irregularly packed and of uniform size. The appearance of the secondary maxima is a criterion of the fibril thickness being highly uniform and of the cross section of the fibrils being circular. As two different methods, slope analysis and peak analysis, made with two different diffraction equipments (the slope analysis with a slit collimated camera with CrKc~ radiation and the peak analysis with a pin-hole collimated camera with CuKc~ radiation) give values that are in close agreement, a fibril diameter of 260 A must be very probable. DISCUSSION The correlation of these results with the body of information already known about the epidermis can only be made on a speculative basis. As mentioned above the electron microscopists have found a diameter of 100 A on the tonofibrils of epidermis. However, the diameter found in this investigation is about 3 times larger for the fibrils of the horny layer. It is therefore probable that several aggregating tonofibrils build up the 260 ~ fibril of the horny layer. One of the main processes in the keratinization of epidermis is perhaps the aggregation of the tonofibrils into the 260 A fibrils. The fact that the 260 A fibrils are scattering independently means that these fibrils are not regularly packed but probably freely movable with respect to each other. A regular packing of the fibrils would possibly lead to a hard and brittle skin as in some dermatoses. An investigation of the molecular packing in some dermatoses is now in progress. It is evident that the molecular arrangement is quite different in the
56
G. SWANBECK
horny layer of the skin and in the hair-cortex. This difference is already present in the germinative epithelium where the epidermis has 100 A tonofibrils and the hair corresponding fibrils of 70 A, which pack together in large bundles of some thousand A. It is very difficult if not impossible to investigate the packing of the peptide chains or the c~-helices in the tonofibrils, because of the irregular arrangement of the fibrils. If the model proposed by Pauling and Corey (25) for the arrangement of the ~helices in s-keratin with six helices twisted around one helix as a seven strand rope is true, it can be assumed that these ropes or protofibrils build up seven strand cables which is the tonofibrils and that the tonofibrils also build up a seven strand cable making a 260 A fibril in the horny layer.
ACKNOWLEDGEMENTS We are grateful for financial support from the research grant No. AF61 (052)-21 from the European Office of Air Research and Development Command, U.S. Air Force, and grant D700, National Institutes of Health, Bethesda, Md., U.S.A.
REFERENCES
l. ASTBURY,W. T. and STREET,A., Phil. Trans. Roy. Soc. London Ser. A 230, 75 (1931). 2. BArm, G., ft'rztl. Forsch. 10, 255 (1956). 3. BEAR, R. S., J. Am. Chem. Soc. 65, 1784 (1943). 4. - ibid. 66, 2043 (1944). 5. BIRBECK, M. S. C., J. Biophys. Biochem. CytoI. 3, 203 (1957). 6. - ibid. 3, 215 (1937). 7. - ibid. 3, 223 (1957). 8. DERKSEN,J. C. and HERINGA,G. C., Polska Gaz. Lekarska 15, 592 (1936). 9. DERKSEN,J. C., HERINGA,G. C. and WEIDINGER,A., Acta Neerl. Morphol. 1, 31 (1937). 10. FRASER, R. D. B. and MACRAE, T. P., Biochim. et Biophys. Acta 29, 229 (1958). 11. FRASER,R. D. B., MACRAE, T. P. and ROGERS, G. E., Nature 183, 592 (1959). 12. GIROU~),A. and CHAMPETIER,G., Bull. soe. chim. biol. 18, 656 (1936). 13. GUINIER,A., Ann. phys. 12, 161 (1939). 14. GUINIER,A., FOtmNET, G., WALKER, C. B. and YUDOW~TC~, K. L., Small Angle Scattering of X-rays. John Wiley & Sons, New York, and Chapman & Hall, London, 1955. 15. KRAT~CY,O. and POROD, G., Z. physik. Chem. N.F. 7, 236 (1956). 16. MAcARTHUR, I., Nature 152, 38 (1943). 17. MATOLTSY,A. G., in MONTAGNA,W. and ELLIS, R. A. (Eds.), The Biology of Hair Growth, p. 135. Academic Press, New York, 1958. 18. MATOLTSY,A. G. and BALSAMO,C. A., J. Biophys. Biochem. CytoL 1, 339 (1955). 19. MATOLTSY,A. G. and ODLAND, G. :F., J. Biophys. Biochem. Cytol. 1, 191 (1955). 20. - J. Invest. DermatoL 26, ]21 (1956). 21. MOBERGER,G. and ENGSTR/3M,A., J. Invest. Dermatol. 22, 477 (1954).
KERATIN FIBRILS OF SKIN
57
22. MONTAGNA,W., The Structure and Function of Skin. Academic Press, New York, 1956. 23. ODLAND, G. F., J. Biophys. Biochem. Cytol. 4, 529 (1958). 24. PAULING, L. and COREY, R. B., Proe. Natl. Acad. Sci. U.S. 37, 205 (1951). 25. - Nature 171, 59 (1953). 26. POROD, G., Acta Phys. Austriaca 2, 255 (1948). 27. - Kolloid-Z. 124, 83 (1951). 28. PORTER, K. R., Anat. Record 118, 433 (1954). 29. ROTHMAN, S., Physiology and Biochemistry of the Skin. University of Chicago Press, Chicago, 1954. 30. RUDALL, K. M., Bioehim. et Biophys. Acta 1, 549 (1947). 31. - Advances in Protein Chem. 7, 253 (1952). 32. SEL~Y, C. C., J. Biophys. Biochem. Cytol. 1, 429 (1955). 33. - J. Soc. Cosmetic Chemists 7, 584 (1956). 34. SWANBECK,G., unpublished. 35. YUDOW~TCH,K. L., J. Appl. Phys. 20, 174 (1949).