Photochemical labeling of membrane hydrophobic core of human erythrocytes using a new photoactivable reagent 2-[3H]diazofluorene.
Photoactivable reagents have been useful for studying the structural aspects of membrane hydrophobic core. We have reported earlier (Anjaneyulu, P.S.R., and Lala, A. K. (1982) FEBS Lett. 146, 165-167) the use of diazofluorene as a probe for fluorescent photochemical labeling of hydrophobic core in artificial membranes. To quantitate and enhance the monitoring ability of this probe, we have synthesized 2-[3H]diazofluorene of high specific activity. This reagent rapidly partitions into phosphatidylcholine vesicles and selectively labels the fatty acyl chains of phosphatidylcholine. The insertion yield (13%) is not affected by the presence of scavengers like reduced glutathione. 2-[3H]Diazofluorene also readily partitions into erythrocyte membranes and on photolysis labels the membrane. The overall insertion was 48% with 9.7% in protein fraction and the rest in lipids. The distribution of radioactivity in labeled protein fraction was restricted to integral membrane proteins with Band 3 being the major protein labeled. There is little or no labeling associated with extrinsic proteins like spectrin. Further analysis of labeled Band 3 by treatment with chymotrypsin indicated that the labeling was restricted to the membrane spanning CH-17 and CH-35 fragments. No labeling of the cytoplasmic fragment of Band 3 could be observed. 2-[3H]Diazofluorene should prove useful for studying integral membrane proteins and their membrane-spanning regions.
D000345:Affinity Labels / Q000378:metabolism*; D002458:Cell Fractionation; D003979:Diazonium Compounds / Q000138:chemical synthesis / Q000378:metabolism*; D004910:Erythrocyte Membrane / Q000378:metabolism / Q000648:ultrastructure*; D005449:Fluorenes / Q000138:chemical synthesis / Q000378:metabolism*; D006801:Humans; D008565:Membrane Proteins / Q000097:blood / Q000302:isolation & purification; D010782:Photolysis; D014316:Tritium
D000345:Affinity Labels; D003979:Diazonium Compounds; D005449:Fluorenes; D008565:Membrane Proteins; D014316:Tritium; C052770:2-diazofluorene