抄録
Trypsin and papain proteinase inhibitors have been identified and purified from aqueous extracts of apple seeds (Malus domestica). Superdex G 75 gel filtration chromatography identified a higher molecular weight (HMW) papain inhibitory fraction (22-26 kDa) and a lower molecular weight papain and trypsin inhibitory fraction (6-12 kDa). The lower molecular weight fraction was separated into a trypsin inhibitor (designated Trpl) and early (designated Papl) and late (designated Pap 2) eluting papain inhibitors after anion exchange (Hitrap SP) chromatography. For Pap 2, two inhibitory peaks (designated Pap 2-1 and Pap 2-2) were identified after further anion exchange (Resource S) chromatography. Each of these lower molecular weight inhibitors was purified by reverse phase HPLC to homogeneity as determined by SDSPAGE and by mass spectrometry. The HMW papain inhibitory fraction was purified further by anion-exchange (Hitrap Q followed by Resource Q) column chromatography where a minor inhibitor (HMWPap 1) and major inhibitor (HMWPap 2) fraction were identified. The relative abundance in seeds of apple and the spectrum of proteinase inhibition has been determined for all of these inhibitors. Reverse-phase HPLC separated HMWPap 2 into a minor (HMWPap 2-1) and a major (HMWPap 2-2) inhibitory fraction, and SDS-PAGE and mass spectrometry confirmed that HMWPap 2-2 was purified to homogeneity. Amino acid composition data were obtained from Trpl, Papl, Pap 2-2, and HMWPap 2-2, and N-terminal sequence data from Trpl, Pap 2-1, Pap 2-2, and HMWPap 2-2, with two of these sequences (Pap 2-2 and HMWPap 2-2) perfectly matching predicted protein sequences based on EST sequences from an apple database. The relationship of these inhibitors with those of other species is discussed.
