Studies on the fluorescence labeling of human red blood cell membrane ghosts with 4'-(9-acridinylamino)methanesulfon-m-anisidide.
4'-(9-Acridinylamino)methanesulfon-m-anisidide (mAMSA) interacts with red cell membranes, resulting in the formation of fluorescent protein adducts. The mAMSA-membrane protein adducts exhibited an emission fluorescence maximum at 445 nm, with two shoulders at approximately 425 and 470 nm. The major labeled proteins were identified as spectrins 1 and 2 and bands 3, 4.1, 4.2 and 5. The fluorescence intensity increased with increasing mAMSA concentrations (0.03 to 1.5 mM), time (15-120 min), and temperature of the reaction. Results from sodium dodecyl sulfate gel electrophoresis show that mAMSA caused no detectable change in the molecular weight of membrane proteins. This indicates that mAMSA is a monofunctional, noncrosslinking agent. Other acridine analogs, 9-aminoacridine and acridine, did not fluorescently label membrane proteins, suggesting that the presence of the acridine nucleus is not sufficient for labeling. Addition of 2-mercaptoethanol to the mAMSA-membrane reaction mixtures reversed the fluorescence labeling. Furthermore, pretreatment of membrane proteins with N-ethylmaleimide or iodoacetamide prevented the formation of fluorescent mAMSA-membrane protein adducts. These data suggest that mAMSA interacts with sulfhydryl groups of the membrane proteins. When the membrane sulfhydryl groups were assayed by labeling with N-[ethyl-2-3H]ethylmaleimide, it was shown that the accessible membrane sulfhydryl groups were reduced after the mAMSA treatment. The above results suggest that mAMSA covalently binds to the sulfhydryl groups in the red cell membrane, with the production of fluorescent mAMSA-protein adducts.
D000609:Aminoacridines / Q000097:blood*; D000677:Amsacrine; D000970:Antineoplastic Agents / Q000097:blood*; D004591:Electrophoresis, Polyacrylamide Gel; D004910:Erythrocyte Membrane / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism*; D008970:Molecular Weight; D013050:Spectrometry, Fluorescence
D000609:Aminoacridines; D000970:Antineoplastic Agents; D008565:Membrane Proteins; D000677:Amsacrine