Charge heterogeneity of recombinant pro-urokinase and urinary urokinase, as revealed by isoelectric focusing in immobilized pH gradients |
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Authors: | Pier Giorgio Righetti Barbara Barzaghi Edoardo Sarubbi Adolfo Soffientini Giovanni Cassani |
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Institution: | Chair of Biochemistry, Faculty of Pharmacy, and Department of Biomedical Sciences and Technologies, University of Milano, Via Celoria 2, Milan 20133 Italy Merrell Dow Research Institute, Lepetit Research Centre, Via R. Lepetit 34, 21040 Gerenzano Varese Italy Tecnogen SpA, Via Ampère 56, Milan 20131 Italy |
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Abstract: | When analysing homogeneous preparations of recombinant pro-urokinase and urinary urokinase by isoelectric focusing (IEF) in immobilized pH gradients, an extreme charge heterogeneity was detected (at least ten major and ten minor bands in the pH range 7–10). This extensive polydispersity was not caused by different degrees of glycosylation, or by IEF artefacts, such as binding to carrier ampholytes or carbamylation by urea. A great part of this heterogeneity could be traced back to the existence of a multitude of protein molecules containing Cys residues at different oxidation levels (-SH, -S-S-, even cysteic acid). Owing to the very large number of Cys residues in pro-urokinase (24 out of a total of 411 amino acids) and to the relatively high pI of its native forms (pI 9.5–9.8; the native form is believed to contain all Cys residues as -S-S- bridges), the presence of SH or cysteic acid residues would increase the negative surface charge, as even SH groups would be extensively ionized. In pro-urokinase, part of the heterogeneity was also due to spontaneous degradation to urokinase and possibly also to cleavage into lower-molecular-mass fragments. When all these causes of heterogeneity were removed, the pI spectrum was reduced to only four, about equally intense, bands. The cause of this residual heterogeneity is unknown. |
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