Identification, characterization, and purification of a 65,000 dalton protein in rat brain that is photolabeled by nitro-containing benzodiazepines

Allen Clarke Bowling, Yale University.

This is an Open Access Thesis


Benzodiazepines bind to two well-characterized classes of nanomolar-affinity binding sites, the central and the peripheral types. Although these sites appear to mediate many of the effects of these compounds, they cannot account for all of the biochemical and physiologic effects of the benzodiazepines. In this investigation, a protein that is photolabeled by NO$\sb2$-containing benzodiazepines was identified and characterized in rat brain by performing photaffinity labeling experiments with ($\sp3$H) -clonazepam and ($\sp3$H) -flunitrazepam. These experiments demonstrate that this photolabeled protein has a molecular weight of 65,000 daltons. Photolabeling of the protein was saturable, inhibited in a stereoselective manner by benzodiazepine enantiomers, inhibited by therapeutically-relevant concentrations of many different NO$\sb2$-containing benzodiazepines, and was not inhibited by more than 70 non-benzodiazepine compounds. The photolabeled protein is distinct from the central and peripheral sites on the basis of molecular weight, benzodiazepine inhibitory potencies, subcellular localization, and tissue distribution.The protein was purified to apparent homogeneity by Affi-Gel Blue and Agarose Green column chromatography. This two-column purification scheme resulted in a greater than 2000-fold enrichment of the protein with a yield of 35%. Biochemical studies on the purified preparation established that the protein exists as a monomer in the purified state, contains a high relative abundance of glycine residues, and possesses amino acid sequence homology with the beta chain of hemoglobin.This newly-characterized protein may mediate some of the effects of the benzodiazepines that are not associated with the central- or peripheral-type sites. It is also possible that the protein is an enzyme that synthesizes or degrades benzodiazepines.