The effect of oxidation or alkylation on the separation of wool keratin proteins by two-dimensional gel electrophoresis.
Comparative two-dimensional electrophoretic (2-DE) studies were performed over a time-course to examine the effect of oxidation or alkylation on the separation of wool keratin proteins. The effect of oxidation was followed by treating scoured wool fibres with increasing levels of hydrogen peroxide, ranging from 0-12 g/L, using conditions mimicking the industrial wool bleaching process. Peroxide treatment was found to have only a minor effect on the 2-DE separation of the intermediate filament protein (IFP) class. Conversely, peroxide treatment of the 24-28 kDa high sulphur protein (HSP) class, which contains up to 40 cysteine residues per protein, resulted in the gradual disappearance of the major HSP spots correlated with the appearance of a few discrete spots at lower isoelectric point (pI). This suggested that only a few specific cysteine residues were being oxidized to cysteic acid by treatment with hydrogen peroxide. Peroxide treatment also appeared to have affected a discrete number of cysteine residues among proteins in the high glycine-tyrosine protein (HGTP) class, reducing the intensity of the high pI spots, while correspondingly increasing the intensity of those at lower pI. In a separate study, wool proteins were alkylated with iodoacetamide (1 M, pH 8) for periods ranging from 10 min to 48 h. In contrast to treatment with peroxide, the pI values of the HSP spots were unaffected by alkylation, irrespective of the length of this treatment. Alkylation resulted in a shift to lower pI and a loss of resolution of individual spots in the Type I and II IFP trains, to the extent that after 24 h alkylation individual spots in these trains merged. In addition after 1 h the intensity of the high pI Type II IFPs decreased until they were no longer visible on the 2-DE map after 24 h. Similarly as alkylation time increased, the major, high pI HGTP spots decreased in intensity. In unison with their decrease, some of the lower pI spots increased in intensity, while new spots appeared at more acidic pIs. Mass spectral studies indicated that cysteine alkylation was relatively fast, with 70-95% of the cysteines in the keratin proteins being alkylated within the first 10 min, while in the case of the HGTPs there was evidence for noncysteine alkylation occurring within this period. Alkylation of proteins for periods of up to 6 h prior to electrofocusing is being promoted as a better alternative to the current 2-DE protocol of the inclusion of a reductant in the immobilized pH gradient rehydration solution. This study has clearly demonstrated that long alkylation times do not suit all protein types or classes.
Plowman
Jeffrey E
JE
Wool Research Organisation of New Zealand, Christchurch, New Zealand. plowman@wronz.org.nz
Flanagan
Leanne M
LM
Paton
Louise N
LN
Fitzgerald
Anna C
AC
Joyce
Nigel I
NI
Bryson
Warren G
WG
D000477:Alkylating Agents / Q000494:pharmacology; D000478:Alkylation; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D003545:Cysteine / Q000737:chemistry; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007460:Iodoacetamide / Q000494:pharmacology; D007526:Isoelectric Point; D007633:Keratins / Q000737:chemistry* / Q000145:classification* / Q000187:drug effects; D007700:Kinetics; D013058:Mass Spectrometry; D008970:Molecular Weight; D016877:Oxidants / Q000494:pharmacology; D010084:Oxidation-Reduction; D012756:Sheep; D014935:Wool / Q000737:chemistry*
D000477:Alkylating Agents; D000596:Amino Acids; D016877:Oxidants; D007633:Keratins; D006861:Hydrogen Peroxide; D003545:Cysteine; D007460:Iodoacetamide