Recovery of the oxidative activity of caged haemoglobin after photolysis

L. Bedouet (1) , Hervé Adenier (1) , S. Pulvin (1) , Catherine Bedel-Cloutour (1) , D. Thomas (1)

1 GEC - Génie Enzymatique et Cellulaire

Caging of bovine haemoglobin with increasing amounts of 1-(2-nitrophenyl)ethyl (NPE) and uncaging after a 366 nm irradiation was examined. Caged and photolysed conjugates were characterised by enzymatic assay of the ABTS oxidation, UV/Vis absorbance, and electrospray mass ionisation. Modification of haemoglobin with 50, 75, and 100 equivalents of 1-(2-nitrophenyl)diazoethane led to a progressive decrease of enzymatic activity. Photolysis at 366 nm during 5, 15, and 30 min induced the recovery of a part of the enzymatic activity. ESI analyses showed that a reversible binding of up to 6 NPE groups per ?-chain and that the removal of most of the photolabile groups occurred rapidly after 5 min of illumination at 366 nm and reached near completion after 15 min. A variable alteration of haemoglobin after labelling could explain that the complete removal of NPE groups did not restore its full oxidative activity.

Mots clés: Hemoprotein; Caged haemoglobin; Photolysis; Chemical modification; Inactivation

Domaines: Chimie

L. Bedouet, Hervé Adenier, S. Pulvin, Catherine Bedel-Cloutour, D. Thomas. Recovery of the oxidative activity of caged haemoglobin after photolysis. Biochemical and Biophysical Research Communications, 2004, 320, pp.939-944. ⟨10.1016/j.bbrc.2004.06.043⟩. ⟨hal-00080299⟩