Synthesis and characterisation of magnetised Dacron-heparin composite employed for antithrombin affinity purification.
Human antithrombin is a blood derivative widely used in the treatment of coagulation dysfunction. Affinity chromatography using heparin (HEP) derivatives is usually used for antithrombin purification. In this study, an affinity procedure based on a magnetic Dacron-HEP composite is proposed. Dacron was firstly converted to Dacron-hydrazide and magnetised by co-precipitation with of Fe2+/Fe3+(mDAC). HEP was activated by carbodiimide and N-hydroxysuccinimide and covalently linked to mDAC (mDAC-HEP). EDX and infrared spectra analyses confirmed each synthesis step of mDAC-HEP. This composite exhibited superparamagnetism behaviour. Human plasma was incubated with mDAC-HEP (fresh and stored over a long period) and washed with phosphate buffer containing increasing concentrations of NaCl. Human plasma antithrombin activity was reduced by approximately 20% in the presence of the 1.0M NaCl fraction, and this eluate was able to prolong coagulation time (aPTT) using both preparations. Electrophoresis of the eluates revealed bands corresponding to the expected size of antithrombin (58kDa). The mDAC-HEP particles are reusable. This method presents the following advantages: easy, low-cost synthesis of the composite, magnet-based affinity purification steps, and reusability.
Laboratório de Imunopatologia Keizo Asami, Universidade Federal de Pernambuco, Cidade Universitária, 50670-901 Recife, PE, Brazil.
Aurenice Arruda Dutra das
AA
Mercês
Laboratório de Imunopatologia Keizo Asami, Universidade Federal de Pernambuco, Cidade Universitária, 50670-901 Recife, PE, Brazil.
Ricardo de Souza
RS
Silva
Departamento de Física, Universidade Federal de Pernambuco, Cidade Universitária, 50670-901 Recife, PE, Brazil.
Karciano José Santos
KJ
Silva
Centro de Ciências da Saúde, Universidade Federal de Roraima, Aeroporto, 69310-000 Boa Vista, RR, Brazil.
Jackeline da Costa
JD
Maciel
Centro de Ciências da Saúde, Universidade Federal do Recôncavo da Bahia, Centro, 44380-000 Cruz das Almas, BA, Brazil.
Givanildo Bezerra
GB
Oliveira
Departamento de Física, Universidade Federal de Pernambuco, Cidade Universitária, 50670-901 Recife, PE, Brazil.
Davian Martinez
DM
Buitrago
Departamento de Física, Universidade Federal de Pernambuco, Cidade Universitária, 50670-901 Recife, PE, Brazil.
José Albino Oliveira
JA
de Aguiar
Laboratório de Imunopatologia Keizo Asami, Universidade Federal de Pernambuco, Cidade Universitária, 50670-901 Recife, PE, Brazil. Electronic address: lbcj.br@gmail.com.
Luiz Bezerra
LB
de Carvalho-Júnior
D000991:Antithrombins / Q000097:blood* / Q000302:isolation & purification*; D002846:Chromatography, Affinity / Q000379:methods*; D006493:Heparin / Q000737:chemistry*; D006801:Humans; D059346:Magnets / Q000737:chemistry*; D010314:Partial Thromboplastin Time; D011093:Polyethylene Terephthalates / Q000737:chemistry*
D000991:Antithrombins; D011093:Polyethylene Terephthalates; D006493:Heparin
Antithrombin; Heparin; Immobilisation; Magnetic support; Purification