Affinity purification of proteinases by a combination of immobilized peptidyl aldehyde and semicarbazone

Abstract

d-Phe-Phe-argininal semicarbazone and Tyr-Gly-Gly-Phe-Leu-Arg-argininal semicarbazone were prepared using the solution phase synthesis method and characterized by mass spectrometry and nuclear magnetic resonance spectroscopy. The tripeptide and heptapeptide semicarbazones were individually immobilized on affi-Gel 15 resulting in two affinity columns called S 3 and S 7, respectively. A third affinity column was obtained by hydrolysing the semicarbazone moiety in column S 3 to aldehyde (column A 3). Serine proteinases such as trypsin or rat plasma kallikrein almost quantitatively bind to either S 3 or A 3 affinity columns. Under optimized conditions, more than 97% of trypsin bound to both columns S 3 and A 3. At a lower ionic strength and higher pH, 80–85% of rat plasma kallikrein bound to the same columns. Elution of both enzymes was achieved using mild conditions at near neutral pH and in the presence of a small amount of denaturant. Both proteinases were identified and characterized by high-performance liquid chromatography, sodium dodecylsulphate polyacrylamide gel electrophoresis and by their substrate specificity and inhibition profiles. A single purification (six- to seven-fold) step using either column S 3 or A 3 allowed the preparation of pure trypsin from commercial sources. Starting from rat plasma partially purified by a phenyl boronate column, fractionation on the S 3 column allowed approximately an 87-fold purification of rat plasma kallikrein. However, serial purification of rat plasma kallikrein on column S 7 followed by column A 3 resulted in a purification factor of about 455.