Reactions of Hemoglobin and Hematin with Sodium in Liquid Ammonia

Abstract

We have shown, that certain proteins and peptones are acidic in liquid ammonia and react with sodium with liberation of hydrogen. Proteins and peptones liberate approximately one-half of a molecular weight of hydrogen for each gram atom of nitrogen in the substance when an excess of sodium is used. Hemoglobin and hematin react profoundly differently toward sodium in liquid ammonia than do the other proteins which we have studied. They liberate hydrogen continuously when excess sodium is present and in quantities disproportionate to the acidic groups present. Their behavior resembles the catalytic action of a rusty iron nail in decomposing the ammonated sodium atom to give sodium ion, amide ion, and hydrogen. The analytical procedure was essentially the same as that described previously. The substances were dried for 48 hours in an electric oven at 80°C. and then kept in a vacuum desiccator over sulphuric acid. Hemoglobin is not appreciably soluble in liquid ammonia. It swells, softens, and the particles tend to coalesce. Within a few moments, the supernatant liquid is yellow, but slowly turns to a reddish color after 20 min. After standing for 4 hours, the solution is a light green. The change in color may be due to impurities present. There is a persistent increase in rate of boiling of liquid ammonia following the addition of hemoglobin suggesting that ammonation of the molecules is taking place exothermieally. When 0.1 gm. of sodium is added to 0.1 gm. of hemoglobin in liquid ammonia, hydrogen is evolved rapidly during the first 15 min. After that the rate was slowed up considerably, but it proceeded until the blue color of the solution was decolorized. If more sodium was then added, the reaction would start again and continue until the solution was decolorized again. This could be repeated several times. The volume of hydrogen evolved was far in excess of the amount liberated by the same weight of silk, zein, egg albumin, casein, and edestin. The rate is characteristic of a substance containing a carboxyl group, as an amino acid. Hemin contains 2 carboxyl groups, and globin probably contains some free carboxyl groups, although the basic groups in it are in excess. It is possible that our method of drying did not remove all of the water. Water, being acidic in liquid ammonia, reacts rapidly with sodium liberating hydrogen. Hofner reports that 100 gm. of hemoglobin contains 9.25 gm. of water of crystallization.