CHARACTERIZATION OF WOOL INTERMEDIATE FILAMENT PROTEINS SEPARATED BY MICROPREPARATIVE 2-DIMENSIONAL ELECTROPHORESIS

Wool intermediate filament proteins (IFP) are a subclass of the cytoke ratins, a group of structural proteins which form intermediate filamen ts in many cell types, Post-translational modifications, such as phosp horylation, play an important role in the control of intermediate fila ment assembly. Two-dimensional electrophoresis has previously been use d to study the IFP distribution in wools with different physical chara cteristics. Charge heterogeneity has been observed in Type I and Type II IFP. In a previous study, two-dimensional electrophoresis of alkali ne phosphatase-treated wool protein extracts was used to show that Typ e II IFP are phosphorylated. To facilitate post-separation analysis, m icropreparative two-dimensional electrophoresis was used to separate m illigram quantities of wool protein. Direct phosphoamino acid analysis has confirmed the presence of phosphorylation on serine residues on T ype II IFP, whose identity was confirmed by amino acid compositional a nalysis, The isoelectric points of Type I IFP are very similar and the y do not separate completely on the commercially available pH 4-7 immo bilized pH gradients (IPG) used in this study, In situ tryptic digesti on followed by automated Edman sequencing of the. high performance liq uid chromatography (HPLC)-separated peptides was used to confirm the i dentity of this group as Type I IFP. To improve the separation of the Type I IFP it will be necessary to use narrow range IPGs such as Immob iline DryPlates which are available from Pharmacia Biotech, in the pH ranges 4.2-4.9, 4.5-5.4, 5,0-6.0 and 5.6-6.6.