516 MATIAS AND ORENTREICH

We are grateful to Dr. A. L. Burlingame and the staff of t he Biomedical Mass Spectrometry Resource for assistance in obtaining the mass spectral data, and to J eannette M. Bonifas and Dr. P. W. Lambert for helpful suggestions and technica l assistance.

REFERENCES 1. S hapiw LJ: Steroid sulfatase defic iency and X- linked ichthyosis,

The Metabolic Basis of Inheri ted Disease, 5th eel. Edited by JB Stanbury, JB Wyngaarden, OS FredJ·ickson, JL Goldste in, MS Brown. New York, McGraw-Hill, 1982, pp 1027-1039

2. Epste in EH Jr, Williams ML, E lias PM: Steroid sul fatase , recessive X- linked ich thyosis and stratum corneum cell cohesion (edito­rial). Arch Dermatol ll7:761-763, 1981

3. Sahashi Y, Suzuki T , Higaki M, Asano T: Metabolism of vitamin D in animals . V. Isolation of vitamin D sulfate from mammalian milk. J Vitaminol 13:33-36, 1967

4. Asano T, Hasegawa T, Suzuki K, Masushige S, Nose T , Suzuki T: Determination of vitamin D:~-sulfate in milk by high performance liquid chJ·omatog1·aphy. Nutr Rep Int 24:451-457, 1981

5. HoLlis BW, Roos BA, Draper HH, Lambert PW: Occurrence of vitamin D sulfate in human milk whey. J Nutr 111:384-390, 1981

6. Nagubandi S, Landowski JM, Bollman S, Tietz P, KumaT R: Synthesis and biological activi ty of vitamin D:~ 3,6-sulfate. Ro le of vitamin D:1 sulfates in calcium homeostasis. J Bioi Chern 256:5536-5539, 1981

7. Reeve LE, DeLuca HF, Schnoes HK: Synthesis and biological activity of vitamin D3-sulfate. J Bioi Chern 256:823- 826, 1981

8. Daculsi G, Kerebel B , Kerebel L: Action de Ia vitamine 0 3 et du

0022-202X/83/8005-0516$02.00/0 T HE JOURNAL o~- ] NVESTIGATI VE D ERM ATOLOGY , 80:5 16-519, 1983 Copyright © 1983 by The Williams & Wilkins Co.

Vol. 80, No. 6

sulfate de D3 sur les tissus denta ires et osseux du pore. J Bioi Buccale 9:363-374, 1981

9. Reeve LE, J orgensen NA, DeLuca HF: Vitamin D compounds in cows' milk. J Nutr 112:667-672, 1982

10. Higaki M, Takahashi M, Suzuki T , Sahashi Y: M etabolic activities of vita min D in animals. III. B iogenesis of vitamin D sulfate in animal tissues. J Vi taminol11:261-265, 1965

11. SjovaU J, Vihko R: Analysis of solvolyzable stero ids in human plasma by combined gas chromatography-mass spectrometry. Acta Endocrinol (Copenh) 57:247-260, 1968

12. Sorgue Y, Miravet L: Chromatographic separation of vitamin D 3 sulfate and vitamin 0 ;, . Steroids 31:653-660, 1978

13. S hackleton CHL, Whi tney JO: Use of Sep-Pa k car tridges for uri­nar y steroid extraction: evaluation of t he method for use prior to gas chromatographic analysis. Clin Chim Acta 107:231-243, 1980

14. Aberth W, Straub K, Bmlingame AL: Secondary ion mass spec­t rometry with cesium ion primary beam and liquid target matrix for ana lysis of bio-organic compounds. Anal Che rn 54:2029- 2034, 1982

15. S hackleton CHL, Straub K: Direc t analysis of stero id conjugates: the use of secondary ion mass spectrometry. Steroids 40:35-51, 1982

16. Epstein EH Jr, Leventha l ME: Steroid sulfatase of human leuko­cytes and epidermis and the diagnosis of recessive X- linked ichthyosis. J Clin Invest 67:1257-1262, 1981

17. S hapiro LJ , Cousins L, Fluhru·ty AL, Stevens RL, Kihara H: Steroid sulfatase deficiency. Pediatr Res 11:894-897, 1977

18. lwamori M, Moser HW, Kishimoto Y: Solu bilization and par t ial purification of steroid sulfatase from rat liver: characterization of estrone sulfatase. Arch Biochem B iophys 174:199-208, 1976

Vol. 80, No. 6 Printed in U.S. A.

Stimulation of Hamster Sebaceous Glands by Epidermal .Growth Factor

JONATHAN R. MATIAS, M.S. AND NORMAN 0RENTREICH, M .D., F.A.C.P.

Orenlreich Foundation for the Advancement of Science, Inc. (JRM), New Yorh, and New Yorh University School of Medicine (NO), New Yorh, New Yorh, U.S.A.

Subcutaneous injection of epidermal growth factor (EGF) into the pinna of adult female and castrated male Syrian hamsters resulted in an increase in the number of cells p er sebaceous gland unit. The effect of EGF on the sebaceous cell number was localized to the treated ear and accompanied by epidermal hyperplasia. The inje ction of testosterone into the ear also produced an increase in the cell number. When testosterone and EGF w ere injected toge ther, the two hormones exerted an additive effect on the sebaceous glands of female ham­sters. This is the first demonstration of a sebotrophic action of this polypeptide hormone.

The size and secretory activity of the sebaceous glands are influenced not only by androgens [1) and other steroids, but also by other nonsteroidal facto rs. In the rat, thyroxine and a ­melanocyte stimulating hormone have been shown to produce significant stimulation of sebaceous gland function [2-4]. Tis­sue-specific mitotic inhibitors, termed chalones, have also been

Manuscript received April 26, 1982; accepted for publication October 26, 1982.

Heprin t t·equests to: J onathan R Matias, Orentreich Foundation for t he Advancement of Science, Inc., 910 F ift h Avenue, New York, New York 10021.

Abbreviations: EGF: epidermal growth factor

proposed as modulators of cell division under normal physio­logic conditions. Bullough and Lawrence [5] have extracted a chalone which inhibits mitosis in the sebaceous glands of the mouse.

Epidermal growth factor (EGF) , a low-molecular-weight polypeptide chain consisting of 53 amino acids, belongs to a group of naturally occurring peptides that stimulate the prolif­eration of diverse cellular systems both in vivo [6-8] and in vitro [9,10]. Induction of epidermal hyperplasia and hyperke­ratinization by EGF when administered in vivo is well docu­mented [7,11]. However, little is known about the effect ofEGF on skin appendages. Since the sebaceous glands ar e epidermally derived structures, we have studied the effect of EGF on seba­ceous glands.

MATERIALS AND METHODS

Anima.ls

Adult, sexually mature Syrian golden ha msters (> 3 months of age, 110-120 g) were obtained from a randomly bred colony main tained in our la borato ry. The animals were housed in groups of 3-4 animals per cage and ma in tained at a constant photoperiod (1 4 h of light, 10 h of darkness). Food (Purina Laboratory Chow) and drinking water were provided ad libi tum.

Chemicals

EGF was purchased from Collaborative Research, Inc. (Waltham, Massachusetts) in lyophilized form and resolubilized with 0.9% saline

J une 1983

to the desired concentrations. T he aqueous suspension of testosterone (Oreton) was obta ined from Schering Corp. (Bloomfield, New Jersey) and dilu ted with o·.9% saline.

Injection and Ear Shin Evaluation

Varying concentrations of EG F or testosterone in 0.1 cc of 0.9% saline was injected subcutaneously in to the right ventra l ear skin through a 30-gauge needle. T he control animals were injected wi th 0.1 cc of 0.9% saline in the right ear. In all an imals the contralate ral ears were un treated and were used as an index of any possible systemic hormonal effects or local traumatic effects of the injection. T he inj ec­tions were given on alte rnate days between 1300 and 1600 hours.

At t he end of each experiment the animals were sacrificed by cervical dislocation. All biopsies were obtained from t he medial section of the pinna and fixed in 10% phosphate-buffe red formalin . The tissues were em b e dded in paraffin wax, sectioned at 7 J.tm thickness, and stained wi t h h ematoxylin and eosin.

Counts were made of the tota l number of cells per sebaceous gland unit seen in cross section. T he ce ll counts consisted of basal cells, sebaceous cells at va rious stages of maturation, and cells extending in to th e sebaceous duct. F rom each section 10- 15 sebaceous gland units were counted and a mean value fo r each animal was calculated .

T hirty females were divided in to 2 groups. Animals in Group I were injected wi th 1 f.!g of EG F on alternate days while those in Group II were injected wi th 0.9% saline. On days 1, 3, 7, 10, and 17 three animals from each group were sacrificed and the num ber of sebaceous cells per glan d was dete rmined. In another experiment, groups of 5 or 6 female hamsters were injected with either EGF or testosterone at various dosages fo r 1 week. Male hamsters were castrated via the scrotal route under ether anesthesia. Four weeks after the operation, the castrated males were injected wi th 5 J.tg of EG F on alternate days fo r 1 week Controls were sham operated and injected with saline.

F orty fe male hamsters were divided in to 4 groups and injected as fo llow s: Group I, 1.0 l'g of EG F; Group II, 2.5 l'g of testosterone; Group Ill, 1.0 J.tg of EG F plus 2.5 f.!g of testoste rone; and Group IV, saline. Inj ections were given fo r 1 week and ear biopsies were obtained on day 7.

Statis tics

Student's t-test was used to evaluate the significance of the data.

RESULTS

Whe n E GF was injected into th e fem ale h a mster pinna, a significant increase in t he cell num ber ( < 0.01) was obse rved as

120 f-

:z :::::> 100

cn c::) _j:z _j4 w_j

~0 (._) (!)

LL 0 (/)

:::::> 0::0 ww roo ~;:i3 ::::>w z (/)

0: w a.. 20

2 4 6 8 10 12 14 16 18

DURATION OF TREATMENT (Days) FIG l. The number of ce lls per sebaceous gland uni t ·from a random

cross section of the eru· pinna of adult female hamsters injected with 1.0 p.g of EGF on alternate days. Each point represents the mean and SEM of 3 animals. e, EGF-treated eru·s; 0 , untreated contralateral eru·s of EGF-treated females; A, saline-treated females; 6, untreated contra­latera l ears of saline-treated females.

E FFECT OF EGF ON SEBACEOUS GL AN DS 517

b

FIG 2. Photomicrographs of the sebaceous glands from a randon1 cross section of the ear pinna of (a) adul t female hamsters injected with l.O f.lg EG F or (b) saline fo r 17 days. Scale = 100 run.

early as day 7 (Fig 1). By day 17 t h e cell number increased by a pproximately 150% in comparison to t h e saline-t reated control a nimals(< 0.01). The unilateral injection of 1.0 11-g o f EGF did not exer t a systemic effect. The cell CO!J nts of t h e untreated ear were s imila r to th e values obtained fo r the saline-injected cont rols.

The photomicrographs of t he sebaceous gla nds (Fig 2) showed that t h e increase in cell number was accom panied by gla ndular enla rgem ent . Epiderm al hyperplasia was qui te evi­dent in t h e EGF-t reated ears. After 17 days of EGF inj ection a 3-fo ld increase in t he t hickness of t h e via ble epidermis can be obser ved in a ll animals. The 2-3 cell layers normally observed in saline-treated and unt reated ears h a d been tr a nsformed in to 8-10 layers of epidermal cells .

S ince t he effect of E GF was statistically significan t after 1 week of t reatm ent, t his t ime period was ch osen to evaluate t he influence of EGF at different dosages. Fig 3 shows t hat a dose­related increase in cell number was obtained on t h e t reated ears. At 2.5 11-g/day th e cell number increased by 127% over t h e control value(< 0.001 ). H owever, increasing th e dose to 5 and 10 11-g/day did not produce fur th er increase in cell number. M easurem ents ma de on t h e con tralateral ears of fem ales in ­jected wi th various doses of EGF sh owed no statistically sig­nificant difference from th at of t h e con trol. This indjcates t hat the effects of E G F , even at higher dosages, were localized only to the t reated ears.

When castrated males were inj ected wit h EGF, t here was a significant increase in t h e cell number (Table 1). T h e injection of t estosterone into t he ears of fem a les also produced a local, but sm aller increase in cell number (Ta ble II) in comparison to th e E GF -tr eated fema les (Fig 3).

T o determine t he relationship between testosterone and E GF , th ese hormones were inj ected simultaneously into t h e female hamster ear. The data in F ig 4 indicate th at t h e number of cells present was s ignificantly greater ( < 0.001) for fe m ales treated simulta neously wit h testosterone and EG F t han wit h fem ales t reated separately wi th eit h er of th e hor mones. W hen a dminis tered togeth er an increase of 126% was observed . T his corresponds approximately to t h e sum of th e increases pro­duced independently by E GF (64%) and testosteron e (53%).

518 MATIAS AND ORENTREICH

LL. OU"J

90

0::6 60 ww (l)U

::2 ~ 50 ::J w zU"J

DOSE FIG 3. T he response of the fe male hamster ear sebaceous glands to

different dosages of EGF. Each point represents the mean and SEM of the cell number per sebaceous gland uni t from random sections of the ear pinna of 6 animals given injections on alternate days for 1 week. e, EGF-treated ear; 0, untreated contralateral ear.

TABLE I. The effect of EGF on the number of cells per sebaceous gland unit from random sections of the ear pinna of castrated male

Syrian hamsters

Treatment

EGF, 5 iJ.g/day Control

" Mean ± SEM.

(n )

5 5

Cell number"

91 ± 10.8 54.8 ± 8.1

p

0.05

TABLE II. The effect of testosterone at various dosages on. the number of cells p er sebaceous gland u.nit from random sections of

the ear pinna of adu.lt Syrian hamster females

Cell number" Dose (J.tg/ day)

Treated Contralatera l

Vehicle 41.8 ± 2.8 37.3 ± 1.6 0.1 43.1 ± 2.3 42.1 ± 1.9 0.5 43.9 ± 2.4 42.1 ± 1.9 1.0 55.3 ± 3.31

' 39.7 ± 2.3 2.5 60.3 ± 3.9'" 38.6 ± 2.7 5.0 59.4 ± 3.6< 39.7 ± 1.3

"Each point represents the mean ± SEM of 5 ani mals. "P < 0.05 in comparison to the vehicle-treated anima ls. c p < 0.01.

100

f-z BO ::::l

cnO ...JZ ...J<l:

IT w-' 60 ot:> U..(f) O::::l

r+ r+ a::8 W o 40 <D<J: ::;: "' r+-::::l W zen

a:: 20 w a..

E G F T ESTOSTERONE EGF+ CONTROL TESTOSTERONE

FIG 4. T he effect of testosterone and EGF on the number of cells per sebaceous gland unit from cross-sections of the ear pinna of female hamsters. Each valu e represents the mean a nd SEM of 10 anima ls given injections on alternate days for 1 week.

Vol. 80, No.6

DISCUSSION

Although the biochemistry and th e mechanism ofEGF action at th e cellular and subcellular level h ave been extensively investigated in vitro, knowledge of the role of EGF in the normal physiology of intact animal systems is limited. A number of effects have been ascribed to EGF in fetal and neonatal a nimals. EGF h as been reported to induce: the acceleration of lung maturation in fetal rabbits [12]; epiderm al hyperplasia in neonatal rats a nd mice [6,7]; inhibition of the follicular growth of the ovary [13]; and retardation of hair growth in the newborn mouse [14]. In adult animals EGF acts not only to stimulate DNA synthesis [15-17], but also influences a number of appar­ently unrelated physiologic processes, such as the suppression of the immune response in mice [18] and th e inhibition of gastric acid secretion in rats and beagles [19].

The skin is a composite organ containing a variety of ap­pendages and cell types that may require th e presence of EGF. As a structure that depends upon continuous mitosis of the basal layer for the replenishment of mature cells, the sebaceous gland appeared to us to be a possible target of EGF. The hamster ear sebaceous gla nd bears a close resemblance to the human sebaceous gland in morphology and tmnover t ime. Therefore, this model was chosen for the current study rather than the flank organ or the preputial gland of the rat a nd mouse [20].

The EGF-mediated increase in th e number of sebaceous gla nd cells described in this communication introduces another factor which may con trol the physiology of the sebaceous glands. When administered together, testosterone a nd EGF appear to exert an additive, rather than synergistic, effect on t he cell number . A similar type of relationship has been ob­served by Thody and Shuster [2] between thyroxine and tes­tosterone in th e sebum secretion of th e rat. Further studies on the influence of EGF on cellular kinetics a nd lipogenesis are necessary to understand the mechanism of EGF action on the sebaceous gland.

Although the sebaceous gla nds are drastically suppressed after combined gonadal-adrenal-pituitary ablation [21] or by potent antia ndrogen therapy [22], the sebaceous glands are miniaturized but remain as intact units. The same is true for animals and humans with testicular feminization syndrome, where small sebaceous glands are present despite the insensi­t ivity of the t issues to circulating androgens. These observations suggest that a basal level of activity is present in the absence of steroidal and/ or hypophyseal stimulation a nd t hat other factors may contribu te to the maintenance of the differentiated state of this appendage.

EGF has been detected in human fluids [23-25] but no studies on its effect on human sebaceous glands have been made.

REFEREN CES 1. Strauss JS, Kligman AM, Pochi PE: The effect of androgens and

estrogens on human sebaceous glands. J Invest Dermatol 39:139-155, 1962

2. Thody AJ , S huster S: A study of the relationship between the thyroid gland and sebum secretion in the rat. J Endocrinol 54:239-244, 1972

3. Thody AJ , Cooper MF, Meddis D, Bowden PE, S huster S: The sebaceous gland response to tr-melanocyte stimulating hormone and testosterone. J Endocrinol 67:18P- 19P, 1975

4. Thody AJ, Shuster S: Contro l of sebaceous gland function in the rat by a-melanocyte stimulating hormone. J Endocrinol 64:503-510, 1975

5. Bu llough WS, Lawrence EB: Chalone cont rol of mi totic activity in sebaceous gla nds. Cell Tissue Kinet 3:291-300, 1970

6. Cohen S: Isolation of a mouse submaxilla ry gland protein acceler­ating incisor eruption and eyelid opening in the new-born animal. J Bioi Chem 237:1555-1562, 1962

7. Cohen S, E Lliot GA: The stimulations of epidermal keratinization by a protein isolated from the submaxillary gland of the mouse. J Invest Dermatol 40:1-5, 1963

8. Sundell H , Serenius FS, Bm·the P, Friedman Z, Kanm·ek KS, Escobedo MB, Orth DN, Stahlman MT: The effect of EGF on fetal lung matura tion. Pediatr Res 9:371, 1975

9. Carpenter G, Cohen S: H uman epidermal growth factor and the

June 1983

10.

11.

12.

13.

14.

15.

16.

17.

proliferation of human fibroblasts. J Cell P hysiol 88:227-237, 1976

Stoker MGP, P igott D, T aylor-Papadimi triou J: Response to epi­dermal growth factor of cul t ured huma n mammary epithe1ia l cells from benign tumors. Nature 264 :764-767, 1976

Birnbaum JE, Sapp TM, Moore JB Jr: Effects of epiderma l growth factor and cyclic nucleotide modulators on epidermal mitosis. J Invest Dermatol 66:313-318, 1976

Catterton WZ, Escobedo MB, Sexson WR, Gray ME, Sunde ll HW, Stahlman MT: Effect of epiderma l growth factor on lung matu­ration in feta l rabbits. P ediatr Res 13:104-108, 1979

L in tern-Moore S, Moore GPM, Panaretto BA, Robertson D: Fol­licular development in the neonatal mouse ovary: effect of epi­dermal growth factor. Acta Endocrinol (Copenh) 96:123- 126, 1981

Moore, GPM, Panarello BA, Robertson D: Effects of epidermal growth factor on hair growth in the mouse. J E ndocrinol 88:293-299, 1981

Butcher NLR, Patel V, Cohen S: Hormonal factors and liver growth. Adv Enzyme Regu l 16:205-213, 1978

Butcher NLR, Patel V: Effects of epiderma l growth fac to r (EGF) a nd pancreatic hormones on growth of rat live r. J Cell Bioi 75: 195a, 1977

Scheving LA , Yeh YC, Tsai TH, Scheving LE: Circadian phase­dependent stimulatory effects of epiderma l growth factor on

0022-202X/ 83/ 8005-05I9$02.00/ 0 THE JouRNAl, o..- I NvF:ST I GATi v~; OEHMATOLOGY. 80:519- 524, 1983 Copyright © 1983 by The Williams & Wilkins Co.

PROTEINS OF HUMAN HAIR AND NAIL 519

deoxyribonucleic acid synthesis in the tongue, esophagus, and tomach of the adult male mouse. E ndocrinology 105:1475-1480,

1979 18. Roberts ML, Freston JA, Reade PC: Suppression of immune re­

sponsiveness by a submandibular salivary gland factor. Immu­nology 30:811-814, 1976

19. Bower JM, Gamble R, Gregory H, Gerring EL, Wilshire IR: Inhi­bition of gastric ac id secretion by epidermal growth factor. Ex­perientia 31:825-826, 1975

20. P lewig G, Luderschmidt C: Hamster ear model fo r sebaceous gla nds. J Invest Dermatol 68:171-176, 1977

21. Lasher N , Lorincz AL, Rothman S: Hormonal effects on sebaceous gla nds in the white rat. IL The effect of the pituitary-adrena l ax is. J Invest Dermatol 22:25-29, 1954

22. Luderschmidt C, Plewig G: Effects of cyproterone acetate and cru·boxylic acid derivatives on the sebaceous glands of the Syrian hamsters. Arch Dermatol Res 258: 185-191, 1977

23. Stm·key RH, Cohen S, Orth DN: Epidermal growth factor: iden ti­fi cation of a new hormone in human urine. Science 189:800-801 , 1975

24. Carpenter G: Epidermal growth factor i a major growth-promoting agent in human milk. Science 210:198-199, 1980

25. Hirata Y, Moore GW, Bertagna C, Orth DN: P lasma concentrations of immunoreactive human epidermal growth factor (urogastrone) in man. J Clin E ndocrinol Metab 50:440-444, 1980

Vol. 80. No.6 Printed in U.S.A.

Characterization of the Proteins of Human Hair and Nail by Electrophoresis

RoBERT C. MARSHALL, B.Sc., PH.D.

CSIRO, Division of Protein Chemistry, Pa.rlwille, Victoria, A ustralia

Low-sulfur a nd high-sulfur prote ins from human hair and nail w er e characte rized by two-dimensional poly­acrylamide gel e lectrophoresis. Apparent molecular weights (estimated by SDS electrophoresis) oflow-s ulfur and high-sulfur proteins were in the ranges 55,500-76,-000 and 26,500-43,000, r espective ly, but these are prob­ably higher than the r eal values (12-30% for low-sulfur, 75-150% for high-s ulfur) because corresponding wool · proteins behave anomalously in SDS e lectrophoresis . Isoelectric points of the low-s ulfur proteins ranged from 4.9-5.4. Six low-sulfur and 7 high-sulfur major prote ins were common to hair and nail from the sam e individual, but each k e ratin contained 1 additional m ajor low-s ulfur component which was not common. Variation, presum­ably of genetic origin, was observed in the low-sulfur and high-sulfur prote ins of h a ir and nail.

Mammalian hard keratins contain a complex mixture of proteins arranged in a filament-matrix structure which is sta­bilized by formation of disulfide bonds [1,2]. B iochemica l stud­ies of woo l proteins have shown that there are 8 filamentous low-s ulfur proteins [2,3] of M,. 45,000-58,000 [ 4], some of which

Man uscript received Ma rch 23. 1982; accepted for publication No­vember 3, 1982.

Reprin t req uests to: Dr. R C. Marshall, CS IHO, Division of Protein Chemistry , 343 l~oya l Parade, Parkville :!052, Victoria, Austra lia.

Abbreviations: IEF: isoe lect ric focusing SDS: sodium dodecyl sulfate

contain microheterogeneity [5]. Interaction studies [6] and amino acid sequencing [7) indicate that t he proteins belong to two famili es. On t he other hand, t he matrix proteins of wool are more heterogeneous, with 40-100 high-sulfur proteins (3-7 families, M ,. 11,000- 30,000) [2,8-10] a nd about 30 high-tyrosine proteins (> 10 families, M,. < 10,000) [11 ,12], alth ough many of these components are in minor amounts. In general the low­su lfur and high-sulfur proteins of human hair and nail resemble those of wool [8,13,14], but the extent of the similarity is not known because t he human hard keratin proteins have not been characterized in as much detail as t he wool proteins [15].

E lectrophoresis may be used to characterize proteins, and for a complex system two-dimensiona l electrophoresis is particu­la rly informative especially when the proteins are separated in the first dimension according to their isoelectric points and in the second dimension according to apparen t M,.s [16). Isoelec­tric focusing (IEF) has been used in studies of S-carboxymeth­ylated low-sulfur [5] a nd high-tyrosine proteins [11] , but high­reso lu t ion isoelectric focusing of S-carboxymethylated high-sul­fur proteins (isoelectric points pH 3-3.5) has been unsuccessful (unpublished resul ts) despite many attempts to generate pH grad ients in 8 M urea around pH 3 with either commercial a mpholytes or appropriate mixtmes of amphoteric compounds [17). It was for this reason t hat other two-dimensional proce­dures [18,19] were developed in our laboratory to characterize keratin proteins.

As part of our study to increase the knowledge of the huma n keratin proteins, t he following aspects are examined in t he present paper: (1) Heterogeneity of the proteins is investigated by two or more two-dimensional electrophoretic procedures.