Denaturation of Hemoglobin.

Abstract

In a recent series of papers by Anson and Mirsky, 1 the claim is made that hemoglobin, in N/20 HCl, is completely denatured in 3 minutes at 0°C. These authors claim, further, that this denaturation may be reversed to the extent of 75% by simply neutralizing the acid added with an equivalent amount of NaOH containing a small amount of NaCN. These experiments on the reversal of hemoglobin denaturation are, in essential details, substantially the same as those published almost 3 years earlier by Wu and Lin. 2 Wu and Lin, however, considered that they were dealing not with true denaturation but with a pseudo-denaturation which was peculiar to hemoglobin and dependent upon its prosthetic group. In order to test these divergent views, we repeated the experiments of Anson and Mirsky at temperatures which would really denature the protein. Instead of insolubility at the iso-electric point, we made use of precipitin reactions against a specific antiserum to follow the course of denaturation. Since true denaturation of a protein results in a gradual diminution of its ability to react with its antiserum, we may expect that any reversal of this denaturation would manifest itself by a return of its precipitinogenic properties.

A 3% solution of ox carbon monoxide hemogloibn, prepared by the method previously described by Boor and Hektoen 3 was used in these experiments. One volume of this solution was treated with 3 volumes of N/15 HCl and, after standing at room temperature for 10 minutes, the brown mixture was placed in a boiling water bath. At stated intervals, 4 cc. of this mixture was removed and neutralized by the addition of 1 cc. N/5 NaOH and 1/20 cc. N. NaCN.