Determination of the enzyme for the fragmentation of the third component of complement using two-dimensional electrophoresis.
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概要
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After the albumin content of human plasma proteins was decreased by the addition of polyethylene glycol, the plasma proteins were separated in non-denaturing isoelectric focusing (IEF) gel. The separated proteins in the IEF gel were digested with one of three enzymes; trypsin, Achromobacter protease I (API) or Staphylococcus aureus V8 protease (V8). Proteins in the gel were boiled in the presence of mercaptoethanol/SDS, and constituent polypeptides of proteins were separated by SDS-PAGE. The electrophoretic pattern of polypeptide spots were compared to that of non-denaturing IEF/SDS-PAGE. The α chain of the third component of complement (C3α) was effectively digested with trypsin, but albumin and IgG were not effectively fragmented with trypsin. On the other hand, albumin was digested with API and V8 protease, but C3α was not effectively fragmented with these enzymes. These results indicate that trypsin is the most effective enzyme for the fragmentation of C3 in the IEF gel.
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