tca precipitation or gel electrophoresis fig1 for tca precipitation tubes were placed in an ice bath and incubated on for 20 minutes ice cold 20 tca was added to each tube protein pellets were separated by centrifugation radioactivity associated with the protein pellet i e bche bound 3a and 3b was measured in a u03b3 counter the recovery of gt 90 radioactivity in the protein pellet containing bche indicated that binding of 3a and 3b to bche is strong similarly treated mouse serum samples were also analyzed using sds page samples were either subjected to mercaptoethanol sh or were left unreduced u2212 sh the u2014 sh panel of fig1 shows the autoradiogram of nonreducing gel the sh panel shows the corresponding samples run under reducing conditions the addition of nonradioactive derivatives to serum and brief incubation before the addition of the radioactive compounds effectively blocked the binding these data clearly point to the stoichiometric strong and long lasting binding of new drugs to bche it is noteworthy that 3b recognizes binding sites in two forms of bche whereas 3a binds only to the monomer mercaptoethanol reduces the dimer to monomer to which both isomers bind the same way fig1 sh the binding site sensitivity to the configuration of the ligand is even more pronounced in the case of 2a and 2b derivatives where the s p diastereomer 2a recognizes bche binding site and the r p diastereomer 2b does not bind to bche at all fig2 shows the interactions of bche from mouse serum with certain prepared derivatives and analyzed on the same gel after 30 minutes incubation with mouse serum diastereomers that bind to bche in mouse serum do not bind to.