308 BIOCHEMICAL GENETICS 



Exclusive of differential filtration, which may also influence separation, it can be 

 deduced from the above formulae that optimal conditions for ion-exchange chromato- 

 graphy should occur in a resin that is in equilibrium between its acid and salt forms 

 near the isoelectric point of the protein. 



Column chromatography has been used for identification of hemoglobin differences 

 and also for isolation of hemoglobin fractions. Boardman and Partridge 112 first 

 demonstrated the separation of sheep and bovine hemoglobins on ion-exchange resins, 

 and Huisman and Prins 612 developed the method for distinguishing several abnormal 

 human hemoglobins. The reviews of Hirs 583 and Moore and Stein 891 should be con- 

 sulted for a complete description of how to assemble a column for chromatographic 

 separations. 



In brief, the components of the system are a reservoir for buffer and a column 

 containing the ion-exchange resin through which the buffer and dissolved protein pass 

 under gravitational force. Use of an automatic fraction collector permits continuous 

 recovery of isolated fractions over periods during which the column need not be attended. 

 The quantity of hemoglobin in the effluent fractions is determined by photometric 

 assay, adsorption at 415, 540, or 575 mu. being commonly used. If the hemoglobins 

 collected occur in different forms, that is, HbO a , HbCO, HbCN, or Hb®, a wave length 

 should be chosen that gives equivalent extinctions for equal amounts of each form of 

 hemoglobin in the mixture. 



Selection of a system of ion-exchange resin, buffer, />H, ionic strength, and elution 

 rate is in part obtained by trial and error. Previous knowledge of the differences 

 between the electrophoretic mobilities or isoelectric points of the hemoglobins to be 

 separated is usually helpful, since the binding or adsorption of the molecule on a resin 

 depends on the existence on the protein of a sufficient charge of sign opposite to that on 

 the adsorbent. In general, there is a relationship between electrophoretic mobility 

 and chromatographic behavior, but exceptions are not uncommon. 610 Amberlite 

 IRC-50 (XE-64 or 97), a carboxylic cation-exchange resin, is most commonly used for 

 separating mammalian hemoglobins. More recently, Huisman et a/. 611 have in- 

 vestigated the use of carboxymethyl-cellulose, which has a higher capacity for the ad- 

 sorption of protein, for separating some human and animal hemoglobins. Many 

 ion-exhange resins have a tendency to bind proteins so tightly that denaturation or 

 alterations of the molecule may occur during chromatography; an appreciable amount 

 of methemoglobin is formed from carbonmonoxyhemoglobin unless chromatography 

 is carried out in the cold at temperatures below 5° C. If proper ventilation is available, 

 KCN can be added to the buffer to convert the methemoglobin formed to cyanmethemo- 

 globin, which has chromatographic properties similar to those of carbonmonoxyhemo- 

 globin. The buffer for ion-exchange chromatography should have the same ionic 

 charge as the adsorbent, to avoid formation of^H variables at the point of interaction 

 of buffer and adsorbent. The pH of the buffer should be chosen so that the protein is 

 actually adsorbed or only a filtration process is accomplished. In general, adsorption 

 of hemoglobin varies inversely with the pW of the buffer. As experimentally de- 



