Kozo Arai

Crosslinking structure of keratin. VI. Number, type, and location of disulfide crosslinkages in low-sulfur protein of wool fiber and their relation to permanent set

Crosslinked structures of the permanent set wool fiber treated with boiling water at a 40% extension state and the control fiber were studied by analyzing the rubberlike force-extension curve of the swollen fiber in a mixed solution composed of equal volumes of 8M LiBr and butyl carbitol. The thiol and disulfide contents of set fibers were also determined. It was found that (1) the disulfide (SS) bonds in low-sulfur (LS) microfibril protein transform into new crosslinkages in boiling water, but the SS bonds in high-sulfur matrix protein remain intact, (2) the SS bonds in α-helical segments becomes reactive only at the extension state of fiber and produces a free thiol group, and (3) intramolecular SS bonds may exist in the α-helical segments. Discussion was also made about the closeness of the number of crosslinkage sites of SS bonds obtained from the present rubber elasticity theory and from the theoretical analysis of the amino acid sequence of the intermediate filament. The cross-linking structure model in LS protein was proposed. It was suggested further that the setting mechanism for new crosslinkage theory seems to be unsatisfactory, since the new crosslinkages do not contribute to stabilize the extended conformation of the wool chain.

Characterization of cross-links introduced in gelatin

A method for the determination of the effectiveness of gelatin hardeners was proposed. Formaldehyde (FA) and 1,3-bis (vinylsulfonyl)-2-propanol (BVSP) were used as cross-linking agents for the hardening of gelatin. The cross-linked gelatin showed a typical rubber elasticity in a swollen state with a mixed solution composed of concentrated aqueous LiBr and diethylene glycol monoalkyl ether. The energy components to total retractive forces were below 0.3. The amount of intermolecular cross-links was estimated by applying an usual rubber elasticity theory for the swollen samples. The number of cross-linking sites was also determined by the amino acid analysis of BVSP cross-linked gelatin. The cross-linking efficiency for the reaction of BVSP occurring in a casting film with less swelling conditions was about four times higher than in a swollen gel. Approximately 92% or more of BVSP used was reacted with the free amino groups of lysine and arginine residues. There was a significant difference between the reactivities of these amino groups. The amino groups of lysine residues were more reactive than those of arginine in a gel state; the reactivities, however, reversed in a thin layer of wet film. The effectiveness of FA was extremely low.

Crosslinking structure of keratin. V. Number and type of crosslinks in microstructures of untreated and potassium cyanide treated human hair

The pattern of the crosslinks in the microstructures of hair was investigated by analyzing the reaction of hair with aqueous KCN by means of chemical and physical methods. It was found that the disulfide (SS) bonds in hair were selectively converted to monosulfide (S) crosslinks by the treatment with 0.08M aqueous KCN solution. The KCN-treated hairs swollen with an 11M LiBr solution containing N-ethylmaleimide showed rubberlike elasticity in a solution composed of equal volumes of 8M LiBr and diethylene glycol monobutyl ether. Stress-strain relations of the swollen hairs were analyzed by applying a rubber elasticity theory. It was demonstrated that: the conversion reactions of SS to S links occur between the low-sulfur (LS) proteins at the initial step of the reaction; SS bond scission between the high-sulfur (HS) proteins commences at a faster rate through the conversion reactions of intermolecular SS bonds to intramolecular S bonds; and the SS bonds within the matrix proteins are less reactive than the intermolecular bonds in the LS proteins. The number and the type of crosslinks in microstructures of the intact hair were also determined. The percentage ratios of the different crosslinks in LS proteins were 27.0% intermolecular SS, 39.0% intermolecular X, and 34.0% intramolecular SS + X links, where X are the crosslinks other than SS links; the values in the HS proteins were 11.9% intermolecular SS and 88.1% intramolecular SS links. The percentages of the number of crosslinks in LS and HS proteins were 13.8 and 86.2% of the total number of crosslinks of hair, 627 μmol/g, respectively.

Grafting onto wool: 10. Quantitative analysis of the high-angle X-ray diffraction intensity from grafted and ungrafted wool keratin

Abstract The equatorial and the meridional scattering intensities at prominent reflections (0.98 nm, 0.465 nm and 0.51 nm) of X-rays by the keratin in grafted fibres stretched in water have been quantitatively determined. Introduction of poly(methyl methacrylate) into the fibres leads to a marked decrease in α-content. It is suggested that the two types of α-helical component present in the wool fibres are identical with respect to the ease of unfolding of the α-helix during fibre extension. During extension, no significant difference was observed for the rates of production of β-materials by native and grafted fibres. On the basis of these experimental findings, the α β transformation of the keratin system is discussed.

Type and location of SS linkages in human hair and their relation to fiber properties in water

The changes in the mechanical properties accompanying the reduction of disulfide (SS) linkages in hair were studied. A variety of extents of thiol groups were introduced into hair by treatments with thioglycolic acid and tri-n-butyl phosphine. The reduced fibers swollen with an aqueous 11M LiBr solution containing N-ethylmaleimide showed typical rubberlike elasticity in a solution composed of equal volumes of 8M LiBr and diethylene glycol mono-n-butyl ether. The crosslink density was determined from the shear modulus of the swollen fiber. It was found that the SS linkages can be divided into two groups : the intermolecular linkages group (SS 1 and SS 2 ) and the intramolecular linkages group (SS 3 ). The effect of the SS bond scission on the shear modulus of the reduced fibers in water was also studied. It was further found that intermolecular SS 1 linkages do not respond to the shear modulus of fiber in water, and the amounts of SS 1 , SS 2 , and SS 3 are about 35, 18, and 47% of the total cystine content in hair (623 μmol/g), respectively. With respect to the location of SS linkages in hair, important suggestions were obtained : the intermolecular SS 1 and SS 2 linkages are located in the microfibril and the matrix, and the former are more accessible to water than the latter ; and SS 3 linkages are localized within the hydrophobic region of the matrix.

Grafting onto wool: 9. Alpha and beta forms in keratin-polymer system

Abstract Various vinyl monomers, methyl methacrylate, methyl acrylate, ethyl acrylate, butyl acrylate, styrene, acrylonitrile, 4-vinyl pyridine, and 2-hydroxyethyl methacrylate were graft-copolymerized onto Lincoln wool fibres. The α- and β-X-ray intensities diffracted from the native and the grafted fibres were analysed quantitatively. by grafting with hydrophobic monomers, the amount of the α-crystallites falls to about one-half of the initial value, but the decrease in α-content is considerably less with hydrophilic monomers. On the other hand, in the grafted wool containing the latter monomers, the production of the β-materials is more less constrained throughout the fibre extension. Results show that the production of the β-materials does not directly correlate with the content of the α-materials remaining in the grafted fibres, and that little possibility exists for the production of the β-cystals from the partly disrupted chains of the α-crystallites resulting from the deposition of polymer.

Grafting onto wool: 7. Determination of end groups of grafted polymer and homopolymer formed in methanol-benzoyl initiating system

Abstract A previous assessment of the end group incorporated in poly(methyl methacrylate) and polystyrene chains, based on the synthesis of the 2,4-dinitrophenyl (DNP) derivatives of hydroxyl and amino groups, has been extended. It has been found that in methanol-benzoyl peroxide initiating systems, hydroxyl, phenyl and benzoyloxy radicals initiate homopolymerization. The total number of end groups has been determined. From the results of the number of DNP-amino-acid end groups in the isolated polymer chain from the grafted fibres and from chemically modified fibres before and after grafting, it is postulated that two DNP-end groups are linked to the polymer chain, and one DNP—amino-acid is situated at each end of the polymer chain. No initiating mechanism visualized with respect to the homopolymerization is involved in the graft copolymerization.

Grafting onto wool: 8. Amino-acid compositions of various fractions of wool graft copolymers synthesised by methanol-benzoyl peroxide system

Abstract The amino-acid composition of the mother protein in the graft copolymer synthesised by the methanol—benzoyl peroxide initiating system was investigated. True graft copolymers were successfully separated from the grafted fibres by applying a fractional precipitation method. Analyses showed that the mother protein contains less cystine, threonine, serine, proline but more lysine, aspartic acid, glutamic acid, alanine and leucine than wool. The amino-acid compositions of any of the fractions obtained were almost identical to that of the low-sulphur S -carboxymethylkerateine protein which is considered to originate in crystalline microfibrils. On the formation of grafting centres in the protein chains in the complex structure of wool, such a selectivity has also been observed for the case of lithium bromide and peroxydisulphate redox initiating system. It was suggested, however, that there are two different types of molecule in the low sulphur protein components involved in these two graft copolymers.

Determination of the number of crosslinks in collagens from mechanical properties of swollen fibers

A method for the determination of crosslink density for collagen fibers was proposed. The number of interchain crosslinkages in whale ligament and rat-tail tendon was estimated by applying an usual rubber elasticity theory. Collagen fibers swollen in a solution composed of equal volumes of 8M LiBr aqueous solution and diethylene glycol monoalkyl ether showed a typical rubber elasticity. The energy components to total retractive forces were similar in order in magnitude for crosslinked natural rubbers, namely, below 0.3. It was found that no intermolecular crosslinkage occurs between tropocollagen molecules in tendon from a 2-month-old rat, while there are about 12 crosslinking sites per molecule in tendon from a 10-month-old rat and 15 sites in whale ligament. The number, type, and crosslinking sites in the tendon crosslinked with 1,3-bis(vinylsulfonyl)-2-propanol is also discussed.

Enthalpies of water in freezing and thawing of wool-water mixtures

Enthalpies of freezing and thawing of water in wool-water mixtures are evaluated by DSC. The two different samples are clean wool extracted with acetone and delipided wool prepared by extraction with a mixed solvent composed of chloroform, ethanol, and water in volume ratios of 19:8:1. On cooling curves at 5 K/min for both samples, there are three main peaks for crystallization at about 228, 240, and 255K. The enthalpy of water corresponding to each peak is determined from the slope of the linear relationship between heat and water content. The enthalpies estimated from the three exotherms are 17, 181, and 287 J/g of water, respectively, for the acetone extracted wool, and the values for the delipided wool are 17, 85, and 281 J/g. On the heating curves, there are two peaks—a broad endotherm beginning at a temperature of T d, which varies with the water content, and a sharp peak due to melting of ice for bulk water at 273K. The respective enthalpies are 222 and 330 J/g for the acetone-extracted wool and 110 and 326 J/g for the delipided wool. A possible explanation is proposed for the marked difference in enthalpy between the samples upon freezing and thawing. Removing the unbound internal lipids increases diffusion resistance to water transport within the fibers. The relationship of Td to water content is also discussed.