The patterns obtained following the electrophoretic separation of the proteins of hair may be of use for identifying or determining the origin of the samples. For this purpose, we have developed a two-dimensional procedure which gives many more protein spots in the electrophoretic pattern than does the one-dimensional method. Although not yet fully tested, it appears that the two-dimensional technique can discriminate between different species and, in some cases, even discriminate between certain individuals within a species. The procedure is extremely sensitive and is capable of producing a pattern from a small part of a single hair fibre. A computer-based scanning analysis of the patterns is needed to utilise the results most efficiently, and first stages in the development are described. We suggest that a large number and wide range of hair samples should be analysed enabling the generation of multi-user data bases for the identification of the origin (species) of a hair sample or the possible discrimination between hair samples from different individuals.

A study has been made of the changes in the constituent proteins of a sample of sulfur-deficient human hair (trichothiodystrophy, TTD) which has a cystine content less than 50% of normal. The constituent low-sulfur proteins, although increased in amount, appear normal (apart from the absence of one minor component) as they contain the same array of subunit polypeptides covering the same range of molecular weights and isoelectric points as normal hair. In contrast, the high-sulfur proteins not only were decreased from the normal 40–45% to less than 10% but the proteins were on average smaller, had a lower than normal cystine content, and had a greatly altered amino acid composition. Fractionation followed by two-dimensional electrophoresis of the fractions revealed that the TTD high-sulfur proteins had lost the large heterogeneous group of ultrahigh-sulfur proteins and at least 8 major high-sulfur protein components but had acquired at least 22 components of lower than normal cystine content, which are either not present in normal human hair or are present at levels too low for detection. It is suggested, on the basis of nutritional studies on sheep and humans, that the loss of the ultrahigh-sulfur proteins is due to the presence of a sulfur-deficiency state, presumably of metabolic origin. The changes in the other high-sulfur proteins may be due to the mutation having affected the operation of a regulatory gene for high-sulfur proteins, as such changes do not occur in wool or hair as a result of dietary manipulation.

The outer shell of translucent keratin has been dissected from the claws of the lizard,Varanus gouldii. It is free of calcium and hydroxyproline, in contrast to the fibrous support, and contains proteins rich in glycine (28 residues %) and half-cystine (13%). These proteins have been obtained in soluble form by treatment with 2-mercaptoethanol in 8M urea at pH 11 followed by alkylation with iodoacetate to giveS-carboxymethyl kerateines. The three major components resolved by SDS polyacrylamide gel electrophoresis have been isolated by fractional precipitation with ammonium sulfate followed by chromatography on DEAE-cellulose or Sephadex. Two of the components, low in tryptophan content, appear to be homologous and are relatively homogeneous with respect to both size and charge whereas the third, a tryptophan-rich material, appears to contain about 20 different molecular species as judged by gel electrophoresis in urea at pH 8.9. The molecular weights of two of the isolated omponents (the tryptophan-rich and the major of the two tryptophanpoor components) are about 13000 as determined by equilibrium ultracentrifugation studies.

Administration of epidermal growth factor (EGF) extracted from mouse submaxillary gland to Merino sheep resulted in a temporary inhibition of the activity of the wool follicles. Subsequently, either complete discontinuities appeared in the fibers resulting in shedding of the entire fleece, or incomplete, in which case the fleece was retained but bore a zone of weakness. The protein composition of the first sample of wool harvested from 1 sheep following infusion for 66 hr with 27.5 mg EGF (0-2 weeks posttreatment) was similar to pretreatment wool. This represented wool fibers which were already present in the follicles at the beginning of infusion. Thereafter, the composition of the wool changed progressively, reaching a maximum divergence from the control in the 3-4 week regrowth period followed by a return to normal by about 10 weeks. Over this period the content of high-sulfur proteins first rose from an initial 19% to a maximum of 30%, then returned to 19%, while the high-tyrosine protein content initially decreased from 12% to 5% and then slowly increased to 12%. In addition to changes in overall protein composition, two-dimensional electrophoresis revealed alterations in the proportions of some individual protein components. These changes were similar to those observed with many other wool growth inhibitors. Smaller doses of EGF (5.8 and 2.9 mg but not 1 mg) had similar effects on wool composition but these were of lower magnitude and there was a delay in reaching a maximum response. Even after 16-18 weeks the wool from these treated sheep differed slightly in composition from the pretreatment samples.

1. Claw keratin of the lizard, Varanus gouldii, is composed mainly of 13,000 mr proteins rich in glycine and cystine. This study deals with a tryptophan-rich group of about 20 constituent proteins comprising about one-third of the mass. 2. As S-carboxymethylkerateines, these proteins were separated by fractional precipitation and gel filtration, then characterized by amino acid analysis, end group analysis, electrophoresis and ultracentrifugation. 3. It is considered that the majority of lizard claw proteins are more closely allied to those of avian beak and claw than feather proteins. 4. Claw keratin also contains minor proteins which resemble mammalian keratin high-tyrosine proteins.

A two-dimensional polyacrylamide gel electrophoretic procedure has been developed to give a very sensitive method which provides a high degree of discrimination between the proteins of different samples of human hair using a sample of 3 mg. Its use is illustrated by examination of hair samples from seven individuals spanning three generations of the one family. Eight areas of variability in the electrophoretic patterns were found each of which corresponds to differences in one or more proteins. In addition to variability in patterns due to genetic variations between individuals, changes caused by cosmetic manipulation and environmental damage to the proteins may also be a useful adjunct for identification. This procedure shows potential for the forensic analysis of small samples, possibly even part of a single fibre, of human hair.

The genetic variability in the proportions of conglutin β and γ components of lupin seed storage proteins was explored in F1, F2 and F3 families generated from a cross between two genotypes of Lupinus angustifolius cv. Uniharvest and a white-seeded selection from CPI 47644. The levels of conglutin γ differed by a factor of two in these parents, but varied continuously in the F2 and F3 families, indicating that the parental difference is due to several genes. These parents also differed in the level of a major conglutin β subunit, with a molecular weight of 30 000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the storage globulins from individual F1, F2 and F3 seeds showed that the high level in cv. Uniharvest is controlled by an incompletely dominant allele, Bsc, at one locus, whereas the low level in CPI 47644 (white-seeded) is due to homozygosity for the recessive allele, bsc. The partial dominance of the Uniharvest gene suggests that the locus has a regulatory or processing function, so it has been designated beta subunit controller. F3 families differed significantly in the level of the conglutin β subunit in Bsc/- seeds, indicating that other loci with smaller effects also influence the proportion of this subunit. The proportion of the β subunit in any seed depends largely on its own genotype, and not on the genotype of the maternal plant. The proportion of conglutin γ in the storage proteins varied inversely with the proportion of the β subunit. The implications of these results for the genetic improvement of the biological value of lupin protein are discussed.

After sheep were defleeced with mimosine, cyclophosphamide or N-[5-(4-aminophenoxy)pentyl]-phthalimide, the first samples of the new growth of wool differed markedly in composition from the pretreatment samples, there being substantial reductions in the high-tyrosine proteins and increases in the high-sulfur proteins. Similar results were obtained with mice dehaired with mimosine and with sheep treated with low levels of mimosine which resulted in weakened wool rather than depilation. The composition of later samples of the regrowth wool showed progressive changes with time. The high-tyrosine proteins tended to approach the pretreatment levels, although this may take up to 12 weeks to occur, whereas the levels of high-sulfur proteins, after the initial increase, often fell below normal. In experiments involving defleecing with cyclophosphamide, the level of the latter proteins was still below normal after 3 months. The possibility that this altered protein composition of keratin fibres is characteristic of that portion of fibre first produced by a new or regenerating follicle was investigated in sheep and mice. It was found that wool follicles regenerating after plucking, and newly operating follicles in young sheep and mice, also produced wool and hair with a reduced content of high-tyrosine proteins. It is suggested, therefore, that the apparent long-term inhibition of the high-tyrosine proteins may not be the direct consequence of the administration of the chemical but rather be characteristic of normal wool and hair regrowth. Infusion of an amino acid mixture lacking methionine into the abomasum of sheep caused the growth of weak wool but did not suppress the synthesis of the high-tyrosine proteins. This is in contrast with previous findings that treatments which weaken wool also suppress high-tyrosine proteins.

The electrophoretic patterns of the keratin proteins from birds' beaks were studied to determine their value as an aid in classification and identification. Beak proteins from 32 individuals, representing 7 orders, 13 families and 26 species of Australian birds, have been compared by electrophoresis on cellulose acetate membranes at pH 8.9. It was concluded that electrophoretic patterns of individuals within a species are identical apart from minor differences in band intensities. Several cases of species-specificity were observed, although birds from different species within a genus did not invariably have dissimilar patterns. Different genera within a family had significantly different patterns, although there was no constant level of difference between related genera. The study was not sufficiently comprehensive to provide taxonomic information, being heavily weighted towards psittacine birds. Nevertheless it appeared that electrophoresis of beak proteins is a more discriminating tool for distinguishing taxonomic differences than is electrophoresis of feather proteins, and might conceivably provide an additional technique for taxonomists.

In the accompanying paper it has been shown that two major groups of proteins (low-sulphur and high-sulphur) of ovine wool, horn, and hoof contain similar components although the overall proportions of the groups of proteins and the relative proportions of components within the groups may show significant differences. In the present paper it has been shown for five other species (echidna, hedgehog, rabbit, ox and man) that the hard keratins produced by one animal contain the same groups of protein components but in different relative proportions. The wide apparent differences in the type and relative proportions of the the low-sulphur components which comprise the major constituent proteins of the microfibrils suggest that microfibrils can tolerate a considerable variation in the constituent proteins and still produce functional structures. The low-sulphur protein components are sufficiently well resolved by sodium dodecyl sulphate-polyacrylamide gel electrophoresis to make this procedure potentially useful for animal identification and classification.

This paper compares the constituent low-sulphur and high-sulphur proteins isolated from the three hard keratins-wool, horn and hoof-produced by the sheep, In general the low-sulphur protein fractions from the three keratins are similar in composition and range of molecular sizes. The same can be said for the high-sulphur protein fractions. However the relative proportions of the component proteins within each fraction vary from keratin to keratin, Hom and hoof are more similar to each other than they are to wool and, compared to wool, they contain less total high-sulphur protein and a smaller proportion of those high-sulphur protein fractions of highest sulphur content. There is a changed distribution of protein components in the low-sulphur protein fractions, and one of the wool components is apparently missing from horn and hoof.

A study has been made of the high-sulphur proteins isolated from keratins to determine the value of using their electrophoretic patterns as an aid in studying the classification of closely related animals. The high-sulphur proteins of 35 different species covering nine orders of the class Mammalia have been examined after electrophoresis in polyacrylamide gels at pH 2.6. From this study it can be concluded that keratin samples which have different electrophoretic patterns can be tentatively judged as coming from animals of different species, although identical patterns do not necessarily mean identity of animal source. The ease of keratin sampling and of electrophoretic analysis could make the method useful as an aid in classification.

The heterogeneity of the reduced and S-carboxymethylated high-sulphur protein fraction from mouse hair has been examined by chromatography and polyacrylamide gel electrophoresis at pH values above and below the isoelectric region. Considerable heterogeneity is observed both in size (molecular weight range 12000-45000) and in charge. Amino acid analysis of a number of column chromatographic fractions shows the high-sulphur proteins to be largely composed of proteins with a carboxymethylcysteine content above 25 residues and a pronounced heterogeneity in arginine content. Their chromatographic behaviour is similar to that observed for the ultra-high-sulphur proteins from wool.

Birds' beaks have an outer shell of hard keratin which consists almost entirely of proteins which are very rich in glycine [about 30 residues per 100 residues (residues %)], contain moderate levels of tyrosine and serine (each about 8 residues %), and which have relatively low contents of cystine (about 2-5 residues %), lysine, histidine, isoleucine and methionine. Major protein fractions in the S-carboxymethyl form isolated from the beaks of six different orders of birds have similar amino acid compositions, isoelectric points (pH 4-2-4-9) and molecular weights (13,000-14,500). Detailed chromatographic electrophoretic and compositional studies of the proteins of kookaburra beak reveal them to be a family of closely related proteins with only limited heterogeneity, in contrast to mammalian keratin systems. The major kookaburra beak fraction is similar in overall composition and molecular weight to fowl epidermal scale, kookaburra claw and turtle scute proteins and shows some resemblance to reptile claw protein. Beaks also contain small amounts of protein which are distinctly different from the major fraction but which resemble feather keratin proteins in composition and size.

Originally set up to assist the textile processing and manufacturing industries, the CSIRO Wool Research Laboratories have contributed significantly to fundamental knowledge of the chemistry and biology of wool.

1. 1. A method is described for the electrophoresis of the high-tyrosine proteins of keratins on cellulose acetate which provides resolution comparable with the previous starch procedure. The new method uses much less protein (5 vs. 50 μg), is much quicker (1 · 5 hr vs. several days) and gives sharper and cleaner patterns than the starch gel procedure. 2. 2. The staining of proteins on cellulose acetate with Coomassie brilliant blue has been improved by the introduction of a one bath fixing and staining procedure together with a single bath for destaining. This now means that the fiftyfold increase in sensitivity of Coomassie blue relative to the commonly used Ponceau S stain can be achieved more easily.

Three treatments known to produce weak wool were imposed on sheep, and the effects on the synthesis of high-tyrosine wool proteins were noted. The treatments were: intravenous infusion of the amino acid mimosine (a potential chemical defleecing agent), intravenous injection of the synthetic steroid Opticortenol (dexamethasone-21-trimethylacetate), and the abomasal infusion of methionine into sheep consuming a diet of wheat. All three treatments caused a partial suppression of high-tyrosine protein synthesis. The inhibition caused by mimosine could not be prevented by the simultaneous infusion of tyrosine or phenylalanine, suggesting that in this system mimosine is not acting as a tyrosine antagonist. The role of phenylalanine in controlling the synthesis of the high-tyrosine proteins in wool was also investigated. Although the infusion of an amino acid mixture minus phenylalanine reduces the level of these proteins, supplements of phenylalanine or tyrosine do not stimulate their synthesis, irrespective of the initial level in the fibre. The improtance of aromatic amino acids in the regulation of the high-tyrosine proteins is therefore uncertain. Suppression of the high-tyrosine proteins is usually accompanied by a stimulation in the synthesis of the ultra-high-sulphur proteins, although there does not seem to be a simple stoichiometric relationship between the two protein types.

The tyrosine content of wool can vary from about 2·3 to 4·4% of the amino acid residues. This variability, which can be attributed mostly to differences in the proportion of high-tyrosine proteins, appears to be partly of genetic and partly of dietary origin, for there are considerable differences in high-tyrosine protein content between the wools from different breeds of sheep and individual sheep of the same breed, and abomasal infusions of zein, maize gluten and wheat gluten strongly repress the synthesis of these proteins. Infusions of an amino acid mixture simulating the composition of zein produced a similar effect to that of zein. No significant reversal of the repression was produced by adding lysine, tryptophan or tyrosine to zein. It is suggested that these inhibitions may result from interference with aromatic amino acid metabolism.

1. 1. The homologous group of mammalian hard keratins show extreme diversity in composition, particularly in cystine, glutamic acid, glycine, leucine, proline and tyrosine. 2. 2. Keratins contain three protein types of strikingly different compositions, their diversity in composition resulting from widely differing contents of these proteins. 3. 3. This has been shown for twenty-four keratins (hair, horn, hoof, claw, quill and baleen) produced by a representative range of mammalian types. 4. 4. As individual keratins attain satisfactory structure-function over a range of protein compositions, the important structural feature of keratins may be the arrangement and cross-linking of these proteins, rather than their actual proportions.