On Determining the Chemical Structure of Proteins 273 



purposes. Nevertheless, despite intensive efforts in many laboratories 

 (cf. Zittle, 5 Moore and Stein, and Porter 7 for references*, only a 

 handful of proteins have been chromatographed successfully. Usually, 

 however, successful chromatography has led to purer products and new 

 information, indicating that if the method could be more widely em- 

 ployed protein chemistry would benefit greatly. 



The difficulties in chromatographing proteins doubtless arise from 

 the fact that proteins are large, fragile, polyvalent molecules. Because 

 of their size, they cannot penetrate into the particles of most column 

 packings the way small molecules do but must be bound largely at 

 the surface. Unless the column packing has a large surface, therefore, 

 the capacity may be so small that effective chromatography is impos- 

 sible.* A limited capacity will also render the behavior of the column 

 very sensitive to variations in the composition of the mixture being 

 chromatographed. Displacement of one protein by another, and com- 

 petition between proteins for a limited number of binding sites, may 

 complicate the interpretation of the effluent curves. Under such cir- 

 cumstances, rechromatography, determinations of enzymatic or other 

 activities in the effluent, electrophoresis, or amino acid analyses of the 

 individual peaks will all prove helpful as ancillary means of following 

 the fractionation and will serve to minimize errors of interpretation. 



The fact that proteins are polyvalent molecules undoubtedly makes 

 effective chromatography more difficult. Most proteins contain several 

 residues of each of the amino acids; hence, no matter what types of 

 linkage bind the protein to the column packing, these linkages are 

 almost sure to be multiple. As has been pointed out before, notably 

 by Tiselius, 9 polyvalent molecules are likely to be all adsorbed or all 

 eluted. The R f is likely to be either one or zero and to change abruptly 

 from one extreme to the other over a rather narrow range of experi- 

 mental conditions. Satisfactory elution analysis, however, requires a 

 reversible distribution of solute between stationary and mobile phases 

 leading to R f values intermediate between zero and one. It has seldom 

 been possible to find such conditions with proteins, and for this reason 

 an eluent of constantly changing pH or ionic strength or both (so- 

 called "gradient elution") has of necessity been employed in several 



* The high capacity for proteins exhibited by XE-64, a ground form of IRC-50, 

 is probably related to its large surface, inasmuch as a bead form of the same 

 resin does not yield satisfactory chromatograms. It is not clear whether the high 

 capacity possessed by the ion-exchange materials made by Sober and Peterson 8 

 from cellulose depends upon the surface of the cellulose or a loose gel structure 

 that permits penetration of protein into the column packing. 



