ABSTRACT
The purification of proteolytic enzymes is usually performed using classical protocols of ion-exchange and size-exclusion chromatography, often coupled to affinity procedures. This chapter describes the procedure ultimately developed for the purification of the tissue and plasma forms of Serine proteinase, and its separation from contaminating proteinases of similar chromatographic properties. This involved the use of three affinity columns, a size-exclusion column and a reversed-phase high-performance liquid chromatography (HPLC) column. This methodology allowed the isolation of a sufficiently pure enzymatic preparation for determination of the NH2-terminal sequence of the two chains of this enzyme. The resulting sequence permitted its identification as plasma kallikrein and the contaminating proteinase as factor XII. The developed procedure exploited both the powerful affinity procedures for selectively purifying proteins and the high resolving power of HPLC for the final purification of both plasma kallikrein and factor XII.
