RP-HPLC peptide mapping methods for the analysis of recombinant human pro-urokinase

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 times, determined as identity indexes. Because of the possible co-elution of some proteolytic fragments, an improvement of the array of information given by the technique can be achieved by setting up a series of experiments under hydrolytic conditions with different enzymes, followed by appropriate RP-HPLC gradient elutions. Such an experimental approach appears to be particularly useful in the examination of proteins with a high molecular weight, where the resulting RP-HPLC maps are complex. Therefore different RP-HPLC peptide mapping methods have been studied for recombinant human pro-urokinase (r-h-proUK), a thrombolytic agent of apparent molecular weight of 46 kD. The RP-HPLC maps indicate that the methods developed are not only suitable for the qualitative control of the amino acid sequence and arrangement of disulphide bonds but also represent the first demonstration of the identity of the primary structure of the recombinant and of the native species, within the limits of the technique.