Affinity purification of proteinases by a combination of immobilized peptidyl aldehyde and semicarbazone.
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 S3 and S7, respectively. A third affinity column was obtained by hydrolysing the semicarbazone moiety in column S3 to aldehyde (column A3). Serine proteinases such as trypsin or rat plasma kallikrein almost quantitatively bind to either S3 or A3 affinity columns. Under optimized conditions, more than 97% of trypsin bound to both columns S3 and A3. 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 S3 or A3 allowed the preparation of pure trypsin from commercial sources. Starting from rat plasma partially purified by a phenyl boronate column, fractionation on the S3 column allowed approximately an 87-fold purification of rat plasma kallikrein. However, serial purification of rat plasma kallikrein on column S7 followed by column A3 resulted in a purification factor of about 455.
Basak
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A
J. A. DeSève Laboratory of Molecular Neuroendocrinology, Clinical Research Institute of Montreal, Quebec, Canada.
Yuan
X W
XW
Seidah
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NG
Chrétien
M
M
Lazure
C
C
D000447:Aldehydes / Q000737:chemistry*; D000595:Amino Acid Sequence; D000818:Animals; D002846:Chromatography, Affinity / Q000379:methods*; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D007610:Kallikreins / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009994:Osmolar Concentration; D010455:Peptides / Q000737:chemistry*; D051381:Rats; D012664:Semicarbazones / Q000737:chemistry*; D014357:Trypsin / Q000302:isolation & purification
D000447:Aldehydes; D010455:Peptides; D012664:Semicarbazones; D010450:Endopeptidases; D007610:Kallikreins; D014357:Trypsin