Recovery of the oxidative activity of caged bovine haemoglobin after UV photolysis.
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 alpha-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.
Bédouet
Laurent
L
Génie Enzymatique et Cellulaire, UMR 6022 C.N.R.S., Université de Technologie de Compiègne, P.O. Box 20529, 60205 Compiègne, France.
Adenier
Hervé
H
Pulvin
Sylviane
S
Bedel-Cloutour
Catherine
C
Thomas
Daniel
D
D000818:Animals; D001391:Azo Compounds / Q000737:chemistry / Q000378:metabolism; D002417:Cattle; D004789:Enzyme Activation / Q000528:radiation effects; D004793:Enzyme Reactivators / Q000737:chemistry / Q000528:radiation effects; D006454:Hemoglobins / Q000737:chemistry* / Q000528:radiation effects*; D007700:Kinetics; D009578:Nitrobenzenes / Q000737:chemistry* / Q000378:metabolism*; D010084:Oxidation-Reduction; D010777:Photochemistry / Q000379:methods*; D010782:Photolysis / Q000528:radiation effects; D011485:Protein Binding / Q000528:radiation effects; D014466:Ultraviolet Rays
C488730:1-(2-nitrophenyl)diazoethane; D001391:Azo Compounds; D004793:Enzyme Reactivators; D006454:Hemoglobins; D009578:Nitrobenzenes