The effect of trinitrophenylation on subunit interactions in L-asparaginase.

L-Asparaginase (L-asparagine amidohydrolase, EC 3.5.1.1) from Escherichia coli B was modified by treatment with 2,4,6-trinitrobenzene-1-sulfonic acid at pH 7.5. The introduction of 13 trinitrophenyl groups into one mol of the tetrameric enzyme (TNP 13-asparaginase) results in a loss of 67% of the catalytic activity while the presence of 20 groups (TNP 20-asparaginase) reduces the enzymatic activity by 88%. The modified proteins are homogeneous as judged by disc gel electrophoresis and by the monodisperse boundary in the analytical ultracentrifuge having a sedimentation coefficient of 7.2 S. The rate of dissociation of the TNP 13-asparaginase is twice as fast and TNP 20-asparaginase three times as fast as that of unmodified asparaginase in 4 M urea. Trinitrophenylated subunits in 8 M urea can reassociate into the tetramer after removal of urea by dialysis or by dilution. hybridization of unmodified and TNP subunits indicates that that trinitrophenyl derivatives qualify as suitable variants for studying subunit interactions in oligomeric proteins.