The hematin-binding reaction as a basis for serum albumin determination.

The heme protein found in plasma in certain diseases associated with excessive hemolysis was shown by Fairley (1) to be the product of a reaction between hematin and albumin. This component has been called methemalbumin. 111 our previous paper we have reported the results of a detailed study of this interaction, using crystallized human albumin and ferriprotoporphyrin IX (2). Each molecule of albumin was found to be capable of reaction with 2 molecules of ferriprotoporphyriu, the product being extremely stable. Association occurred in solutions more alkaline than pH i, while dissoc+iation was favored in more acid solutions. The rea&ou was iudepeudend of ionic strength within the range studied. The product of the reaction exhibited a sharp peak in optical extinction at the wave-length 403 rnp and this suggested employment of the hematin-binding reaction as a meaus of calorimetric determination of albumins in solution. The simple stoichiometry, high association constant, and rapidity of combination were, in addition, favorable characteristics of the reaction. It was noted that albumiu alone, of the plasma proteins studied, interacted with ferriprotoporphyrin in this way. A non-specific enhancement of spectral absorption was observed with other proteins, but this effect was slight compared to the pronounced spectral chauge associated with the albumin-hematiu interaction. Evidence is presented in this paper that the binding is selective for albumin among the plasma proteins and that there is little interference on the part of other components of plasma. Greater specificity could be attained by the use of preliminary fractionation to con-