Oriented immobilization of Thioredoxin on grafted porous polymers

CARBAJAL, M.L.; GARCÍA M. N.; POSKUS, E.; SMOLKO, E.E; GRASSELLI, M.

Villa Carlos Paz, Córdoba, Argentina.

Congreso; XXXVIII Reunión Anual de la Sociedad Argentina de Investigación Bioquímica y Biología Molecular (SAIBBM).; 2002

Sociedad Argentina de Investigación Bioquímica y Biología Molecular (SAIBBM).

Polymeric materials with specific affinity to thioredoxin (Trx) and Trx-fusion proteins will be useful in the development of new supports for protein recognition and solid-state diagnostic kits. The aim of this work was the attachment of the recombinant histidin-patch Trx (HP-rTrx) onto polysulfone (PSf) macroporous materials in two different orientations.Glycidyl methacrylate (GMA) was grafted by mutual radiation polymerisation to activate the PSf. Highly reproducible amount of grafting, ranging between 10 to 100% was obtained by choosing an initial favourable monomer concentration and gamma radiation doses around 6 kGy. Iminodiacetic acid (IDA) and amino phenyl arsine oxide (APAO) were then covalently attached to the grafted polyGMA, yielding 35 mg IDA/ml and 0,4 mg APAO/ml membrane respectively.Later on, HP-rTrx was immobilised onto this surface by two different pathways. The first one involves a complex IDA-Ni2+ and three HP-rTrx´s histidines. The second involves a co-ordination site between APAO and two proximal HP-rTrx´s cysteines, which also corresponds to the active site of the enzyme. The occurrence of the HP-rTrx on the polymers was detected by polyclonal antibody recognition in both materials. Orientation was deduced from the lack HP-rTrx activity on APAO-material.