Interaction between hemoglobin subunits in the hemoglobin . haptoglobin complex.

The accessibility of hemoglobin sulfhydryl groups to modification by p-mercuribenzoate @"B) was tested in order to investigate the effect of the hemoglobinhaptoglobin association on the contact between a and /3 chain subunits.Previous evidence indicates that a molecule of tetrameric hemoglobin binds to haptoglobin as two separate a& dimers.Modification of the hemoglobin a and B chain sullhydryl groups (al04, P93, and p112) by p-MB prevents the formation of a, /3 hemoglobin dimers; nevertheless, a combined mixture of toglobin.Does an a& contact between the hemoglobin a and / 3 chains still exist in hemoglobin.haptoglobin complex, or does the association with haptoglobin cause this contact to come apart?In this study, titration of hemoglobin-haptoglobin complex with p-MB demonstrated an inaccessibility of the reagent to hemoglobin a104 and f i l l 2 sulfhydryl groups which are located in the alpl contact, even under conditions where they react completely in free Mb.This result indicates that formation of complex confers extra stability to an a&like contact beyond that which exists in free hemoglobin.It suggests that the strong binding of the aP"" + /3p"B mixture to haptoglobin accommodates the formation of an otherwise unfavorable a,/? dimer contact.Based on this conclusion, a minimum value of 1 0 ' ' is estimated for the association constant between hemoglobin and haptoglobin. aP-MB and 8""" chains still binds irreversibly to hap-Haptoglobin is a glycoprotein that is found in the an-globulin fraction of most mammalian species and is characterized by its strong binding affinity to hemoglobin.The binding between haptoglobin (Hp) and hemoglobin (Hb) is an example of an irreversible yet noncovalent interaction between two different proteins.The extreme stability of this association indicates a very extended region of contact between the molecules.A wide variety of evidence now suggests that the binding of Hbl to Hp occurs through an a$ dimer of Hb (1-5).In particular, the kinetic studies of Nagel and Gibson (6) indicate that tetrameric Hb is incapable of binding Hp and that its dissociation into alpl dimers is a prerequisite and, in fact, the rate-limiting step for binding.The result implicates the a&