Specific modification of the alpha chain C-terminal carboxyl group of hemoglobin by trypsin-catalyzed hydrazinolysis.

In human deoxyhemoglobin a salt bridge links the alpha carboxyl of Arg-141 of each alpha chain to the epsilon-amino group of Lys-127 of the opposite alpha chain. These salt bridges are believed to contribute to the constraints in the quaternary deoxy (T) structure that lower its oxygen affinity. We have tested this hypothesis by incubating hemoglobin with 2 M hydrazine and trypsin which catalyzes specifically the reversible hydrazinolysis of the alpha carboxyl of Arg-141alpha. X-ray analysis shows the major structural difference between native deoxyhemoglobin and hydrazide deoxyhemoglobin to be the loss of the Arg-141alpha1-Lys-127alpha2 salt bridge and its Arg-141alpha2-Lys-127alpha1 counterpart. Accurate oxygen equilibrium curves of hydrazide hemoglobin show that blocking of the salt bridge has raised the oxygen affinity of the T structure while leaving that of the quaternary oxy (R) structure unchanged.