Immobilized Zinc Affinity Chromatography of Pectin Hydroxamic Acids for Purification of Trypsin Inhibitors from Soybean and Sweet Potato

Abstract

Commercial pectin (with a 94% degree of esterification, DE94) suspended in methanol was reacted with methanolic alkaline hydroxylamine at room temperature for 20 h to prepare pectin hydroxamic acids (PHAs). The prepared PHA was coupled to the epoxy-activated Sepharose 6B gel to get immobilized PHA resins. The immobilized PHA resin was then balanced in column with 2 mM ZnCl2 in 50 mM Tris-HCl buffer (pH 7.9) to test the immobilized Zn-PHA gel as solid phase for immobilized metal affinity chromatography for the purification of trypsin inhibitors (TIs) from soybean and sweet potato. Using TI activity staining, it was found that purified TIs from the commercial soybean and sweet potato after trypsin affinity column purification could be adsorbed onto an immobilized Zn-PHA affinity column and eluted by 100 mM EDTA in 10 mM Tris-HCl buffer (pH 7.9). The immobilized Zn-PHA affinity column was used for TI purifications from crude extracts of sweet potato. The recovery of TI activity for one step was 90%, with 19.74-fold purification increase.

Cromatografia de afinidade por íons metálicos imobilizados (IMAC) de biomoléculas: aspectos fundamentais e aplicações tecnológicas

“…37,49,[57][58][59][60][61][62][63] A literatura mostra que, além destas aplicações, IMAC foi utilizada para separação de diferentes tipos de células microbianas, uma vez que estas, quando possuíam resíduos de histidina acessíveis, apresentavam a capacidade de serem adsorvidas em uma coluna monolítica de estrutura macroporosa composta por criogéis de poliacrilamida com IDA-Cu 2+ imobilizado. 65 Proteínas que contêm naturalmente resíduos de histidinas acessíveis (recombinantes ou não) e apresentam importância comercial no campo terapêutico e diagnóstico, como proteínas do soro humano, 29 imunoglobulinas, 31,35,38,[66][67][68][69][70][71] interferon, 72 proteína C humana, 73,74 fator IX 73,75 e hormônio de crescimento humano, 76 foram purificadas por IMAC utilizando os metais de transição Cu 2+ , Ni 2+ , Zn 2+ , Fe 3+ e Co 2+ imobilizados.…”

Physico-Chemical and Antifungal Properties of a Trypsin Inhibitor from the Roots of Pseudostellaria heterophylla

“…Metal ions could induce changes of proteins in the conformation and thus affect the function and structure of the proteins [ 28 ]. The effects of metal ions on the trypsin inhibitory activity of PHTI were studied in the presence of different metal ions.…”