RP-HPLC PEPTIDE-MAPPING METHODS FOR THE ANALYSIS OF RECOMBINANT HUMANPROUROKINASE

Among the techniques available for the detection of protein structure variants such as single point mutations, RP-HPLC peptide mapping plays a key role owing to the high reproducibility of peptide retention tim es, determined as identity indexes. Because of the possible co-elution of some proteolytic fragments, an improvement of the array of informa tion given by the technique can be achieved by setting up a series of experiments under hydrolytic conditions with different enzymes, follow ed by appropriate RP-HPLC gradient elutions. Such an experimental appr oach appears to be particularly useful in the examination of proteins with a high molecular weight, where the resulting RP-HPLC maps are com plex. Therefore different RP-HPLC peptide mapping methods have been st udied for recombinant human prourokinase (r-h-proUK), a thrombolytic a gent of apparent molecular weight of 46 kD. The RP-HPLC maps indicate that the methods developed are not only suitable for the qualitative c ontrol of the amino acid sequence and arrangement of disulphide bonds but also represent the first demonstration of the identity of the prim ary structure of the recombinant and of the native species, within the limits of the technique.