Denaturation of Hemoglobin.

Abstract

The present study arose from an inquiry into the question: Is the denaturation of hemoglobin reversible? According to general experience denaturation of proteins is irreversible, but reversion of denatured hemoglobin has been reported by Anson and Mirsky. 1 , 2

These workers found that when an alkaline solution of hemochromogen was neutralized, a small amount of hemoglobin was obtained. On the basis of this observation they concluded that there is a reversible equilibrium between hemochromogen and hemoglobin:

and that the position of the equilibrium depends on hydrogen ion concentration. Adding acid or alkali to hemoglobin converts it into hemochromogen, while neutralizing hemochromogen turns it into hemoglobin.

The term hemochromogen is used by these authors to designate, not the reduced iron pyrrol complex which they called hem, but a compound of this substance with globin. Since the solubility of hemochromogen is similar to that of denatured proteins, and since it is formed under conditions which would cause denaturation of proteins, they considered hemochromogen as denatured hemoglobin.

The work of Anson and Mirsky was entirely qualitative, and they stated that the evidence for hemochromogen equilibrium was not conclusive.

We denatured different forms of hemoglobin with acid, alkali, heat and alcohol. We allowed the products so obtained to revert in faintly alkaline solution in which they were soluble. Our work definitely establishes the reversibility of the hemoglobin-hemochromogen equilibrium, but our interpretation differs from that of Anson and Mirsky.

Our findings are as follows:

1 Reduced hemoglobin when treated for a short time with dilute HCl is completely precipitated on neutralization. The precipitate, when dissolved in faintly alkaline solution, reverts to hemoglobin. Under suitable conditions the reversion is practically quantitative. If the acid-reduced hemoglobin solution is exposed to the air before it is allowed to revert, no reversion is obtained. If the exposed solution is reduced with hydrosulphite, reversion can again occur.