The use of metal chelate affinity chromatography on the isolation of Leishmania chagasi promastigote hydrophobic proteinases.
In this work, we have assessed the possibility of isolating metalloproteinase fractions from infective Leishmania chagasi promastigotes. Our strategy was the association of the Triton X-114 method with iminodiacetic chromatography enriched with Zn2+. Thus, by using acid conditions, it was possible to isolate two fractions containing two polypeptides, 59 and 63 kDa. The enzymatic activity assay indicated that the two fractions and the two polypeptides had proteinase activities. In addition, it was proposed that those proteinase activities were affected by the presence of 1,10-phenanthroline, a metalloproteinase inhibitor. With this gentle chromatography strategy proposed it is possible to obtain metalloproteinases from L. chagasi in folding that preserve the enzyme activity. This is important for further studies on pathological complications observed in visceral leishmaniasis.
Alves
J L
JL
Laboratório de Biologia Molecular e Doenças Endêmicas, Departamento de Bioquímica e Biologia Molecular, Instituto Oswaldo Cruz, FIOCRUZ, Av. Brasil, 4365 Manguinhos, CP 926, 21045-900 Rio de Janeiro, Brazil.
Mendonça-Lima
F W
FW
Alves
C R
CR
D000818:Animals; D002846:Chromatography, Affinity / Q000379:methods*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D057927:Hydrophobic and Hydrophilic Interactions; D007891:Leishmania / Q000201:enzymology*; D045726:Metalloproteases / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D010618:Phenanthrolines / Q000494:pharmacology; D011092:Polyethylene Glycols / Q000737:chemistry; D015032:Zinc / Q000737:chemistry
D015853:Cysteine Proteinase Inhibitors; D010618:Phenanthrolines; D011092:Polyethylene Glycols; D045726:Metalloproteases; D015032:Zinc