Preparation of Globin.

Abstract

Schulz 1 in 1898 prepared globin, the protein moiety of hemoglobin, by extracting an acidified solution of oxyhemoglobin with a mixture of ether and alcohol. Most of the pigment went into the ether-alcohol layer, leaving the globin in the aqueous layer. Upon neutralization with ammonia the globin was precipitated which was redissolved in dilute acetic acid and dialyzed. This method is very convenient, but the globin thus obtained is not natural, but denatured, globin, since the ether and the alcohol rapidly denature the protein in the presence of the acid.

Hill and Holden 2 have recently succeeded in preparing some globin which is soluble in neutral solution and which may be natural globin. They did not start with pure hemoglobin, but with washed cells. They observed that the stroma proteins, when swollen with ether, had a great power of absorbing hematin, and they utilized this property for the removal of the pigment from the globin. Cell solution was acidified with HCl and shaken with ether and kieselguhr. The mixture was filtered, and the filtrate was neutralized with ammonia to precipitate the denatured globin. This was again filtered, leaving a soluble globin in the filtrate. To minimize denaturation, all these operations were carried out at —2°.

As far as the separation of the pigment from the globin is concerned, the extraction method cannot be surpassed. The stroma proteins remove only a part of the pigment, the remainder being carried down by the denatured globin. Hill and Holden stated that the globin prepared by their method contained up to about 2% of methemoglobin. It would seem that the globin solution may be contaminated also by the stroma proteins. Working with small amounts of cells and “with scrupulous attention to details,” Hill and Holden reported a yield of globin amounting to about 40% of the original hemoglobin.