Characteristics of fibrous protein and the disulphide cross-linking bond in human stratum corneum.
Fibrous proteins of human stratum corneum were isolated with urea-Tris buffer (pH 9.0) with and without 2-mercaptoethanol (2ME). Comparative studies were made of the biochemical and morphological properties of purified samples obtained with and without 2ME. The final yield of fibrous protein extracted with 2ME was 17 times higher than the yield extracted without 2ME. Identical results were found for the 2 samples by electron microscopic observation, SDS gel electrophoresis with urea and 2ME, and amino acid analysis. However, high molecular weight staining patterns appeared on the SDS gel when the fibrous protein extracted without 2ME was treated with sample buffer containing 4 M urea but no 2ME. These staining patterns were indicative of cross-linking with disulphide bonds. Extraction of the structural proteins with or without 2ME produced the same amount of polymerized fibrous protein. These results suggested that the structural polypeptides of fibrous protein were constructed through ionic forces rather than disulphide bonds. Electron microscopic observation also did not show any differences in the fibrous structures constructed with or without 2ME. The structural polypeptide molecules, than, may be stabilized by partial cross-linking with disulphide bonds. It is possible that the addition of 2ME cleaves disulphide bonds in cell membrane structures and releases fibrous components more effectively than the addition of agents which physically disrupt membrane structures. Consequently, the increase of yield of fibrous protein by the addition of 2ME might be mainly due to cleaving of the membrane structures and partially due to cleaving interpolypeptide disulphide bonds.
D000596:Amino Acids / Q000032:analysis; D004220:Disulfides / Q000032:analysis; D004817:Epidermis / Q000032:analysis* / Q000648:ultrastructure; D006801:Humans; D007633:Keratins / Q000378:metabolism*; D008623:Mercaptoethanol; D008970:Molecular Weight; D010084:Oxidation-Reduction; D011498:Protein Precursors / Q000378:metabolism; D012995:Solubility; D014508:Urea
D000596:Amino Acids; D004220:Disulfides; D011498:Protein Precursors; C004818:prekeratin; D008623:Mercaptoethanol; D007633:Keratins; D014508:Urea