An Electrophoretic Study on "Component 2" Extracted From Wool With Alkaline Thioglycollate

Self Cite

Wool and many mammalian keratins consist of two classes of proteins, one which is higher in sulphur content than the parent keratin and is of basic character in the unmodified keratin and one which is lower in sulphur content and is acidic in character (Alexander and Earland 1950;Corfield, Robson, and Skinner 1958;Gillespie and Simmonds 1960). In the intact fibre the low-sulphur proteins are thought to occur in the microfibrils and the high-sulphur proteins in the matrix, and the two are probably linked together by disulphide bonds (Birbeck and Mercer 1957; Rogers 1959).After the disulphide bonds have been broken, by reduction followed by alkylation with iodoacetate, or by oxidation, the two classes of protein can be separated in two ways. The high-sulphur component may be preferentially extracted in the presence of salt (Corfield, Robson, and Skinner 1958;Gillespie 1962) or of certain specific ions (e.g. zinc or cetyltrimethylammonium ion) which suppress the solubility of the low-sulphur proteins (Gillespie 1962). Alternatively a mixture of the proteins can be extracted at low ionic strength and the lowsulphur proteins separated from the others by precipitation at acid pH values. It. has recently been observed that the latter separation when applied to the reduced and S-carboxymethylated proteins can be complicated by co-precipitation of the two classes of proteins. This paper presents an account of this phenomenon and a summary of conditions suitable for separating mixtures of high-and low-sulphur proteins. Results and Discussion(i) Reduced and Oarboxymethylated Proteins.-Erratic and variable yields of high-sulphur protein were obtained when the low-sulphur proteins were removed from mixtures by precipitation at pH 4·1 at an ionic strength of 0 ,1. It. has been reported that a number of reprecipitations were necessary to remove the highsulphur proteins completely from the low-sulphur protein precipitated under these conditions (Gillespie 1960). A study of artificially prepared mixtures of high-and low-sulphur proteins showed that, as the ratio of low-sulphur protein to high in the mixture increases (Fig. I), so the recovery of high-sulphur protein after precipitation falls. Treatment of the supernatant with Zn2+ at pH 6 (Gillespie 1957) showed that the low-sulphur proteins were always quantitatively precipitated.Further data on this co-precipitation of high-and low-sulphur proteins are shown in Table 1. The mixed high-and low-sulphur proteins used in this experiment were prepared from Merino wool, either by extraction with 0 ·IM potassium * Manuscript

Section: Resultsmentioning

confidence: 99%

The Interaction Between High- and Low-Sulphur Proteins Extracted from -Keratin

Gillespie¹,

O'donnell²,

Thompson³

1962

Self Cite

“…As a result of the fractionation studies of Gillespie and Lennox (1955) and of Gillespie (1960) and of the chromatographic and peptide-mapping studies of O'Donnell and Thompson (1961) and Thompson and O'Donnell (1962) there is a growing realization that the protein of low-sulphur content isolated from wool may not be homogeneous. The evidence from the study of wool by X-ray and electron-microscopy techniques suggests that there is considerable regularity in the structure of the microfibrils, but the recent studies of Filshie and Rogers (1961) and Fraser, MacRae, and Rogers (1962) have shown that these structural elements (generally regarded as the location of the low-sulphur proteins) probably consist of 11 protofibrils.…”

Section: Introductionmentioning

confidence: 99%

The Isolation and Properties of Some Soluble Proteins from Wool III. The Heterogeneity of the Low-Sulphur Wool Protein Scmka2

Gillespie¹

1962

Self Cite

SummaryThe low-sulphur wool protein SCMKA2, which gives only single peaks on electrophoresis and ultracentrifugation over a wide range of pH and concentration, has been divided arbitrarily by ammonium sulphate precipitation into two fractions. Amino acid analysis has shown significant differences between the fractions in some amino acid residues, particularly of S-carboxymethyl cysteine, glycine, phenylalanine, tyrosine, and valine. Both fractions have been shown to be heterogeneous by chromatography on DEAE-cellulose and elution with buffer containing 8M urea. One fraction, that which is less soluble in ammonium sulphate, has the lowest sulphur content of any wool protein so far separated. From this fraction a subfraction was prepared by column chromatography which differed significantly in amino acid composition from either of the ammonium sulphate fractions. These data are consistent with the hypothesis that there are a number of low-sulphur proteins in wool.

“…In the absence of reagents such as urea or detergent the wool proteins in the -SH form are only extractable at pH values where ionization of the thiol groups occurs (Goddard and Michaelis 1935;Gillespie and Lennox 1955). By extraction at pH values of 10,5-11 of the wool reduced at pH 5 it is possible to extract the protein in the thiol form and subsequently this can be rapidly alkylated.…”

Section: (G) Chromatography Of Low-sulphur Wool Protein (Scmka)mentioning

confidence: 99%

“…At pH 4·5 and at 35°C increase of the thiol concentration from 0·5 to 2M gave little or no increase in the extent of reduction. The use of thioglycollate solutions at pH values of 10·5-12·3 (Goddard and Michaelis 1935) for the extraction of wool proteins has been extensively studied in these Laboratories (Gillespie and Lennox 1955;Gillespie 1960). At pH values near 2, Savige (1960) has shown that thiols in the absence of salts can extract considerable amounts of protein and at the elevated temperatures used some lanthionine was formed.…”

mentioning

confidence: 99%

Studies on Reduced Wool I. The Extent of Reduotion of Wool with Inoreasing Conoentrations of Thiol, and the Extraotion of Proteins from Reduoed and Alkylated Wool

Thompson¹,

O'donnell²

1962

SummaryThe reduction of wool with mercaptoethanol and thioglycollate at pH 5 has been compared by measuring the disulphide contents of the reduced wool after alkylation of the thiol groups with iodoacetate at pH 8·5--9. Mercaptoethanol and thioglycollate are very similar in their extent of reduction of wool at low concentrations of thiol but the neutral thiol is more effective as a reducing agent at high concentrations. In contrast with the earlier findings of Patterson et al. (1941) the reduction was driven towards completion with increasing concentrations of thiol as would normally be expected for a reversible reaction. With 4)1[ mercaptoethanol 4% of the disulphide bonds were intact in the reduced wool but the extracted proteins contained no disulphide groups. The limit of extractability of reduced wool prepared in this way, and subsequently alkylated with iodoacetate, was about 75%. INTRODUOTIONIn the early studies on the reduction of the disulphide bonds in wool at pH values above 6. At pH 4·5 and at 35°C increase of the thiol concentration from 0·5 to 2M gave little or no increase in the extent of reduction. The use of thioglycollate solutions at pH values of 10·5-12·3 (Goddard and Michaelis 1935) for the extraction of wool proteins has been extensively studied in these Laboratories (Gillespie and Lennox 1955;Gillespie 1960). At pH values near 2, Savige (1960) has shown that thiols in the absence of salts can extract considerable amounts of protein and at the elevated temperatures used some lanthionine was formed. Since in general the rate of lanthionine formation increases markedly with both pH and temperature (Cuthbertson and Phillips 1945;Zahn, Kunitz, and Hildebrand 1960), the use of high pH values at elevated temperatures for the reduction and simultaneous extraction of proteins of wool is, from this point of view, less satisfactory than reduction at neutral pH values at room temperature followed by extraction of reduced proteins. At pH values of 11 (or higher) at -room temperature we have found that there is an increase in ninhydrin colour after 24 hr with reduced and alkylated proteins (O'Donnell and Thompson 1962) which could be indicative of peptide bond hydrolysis.