INTERACTION OF ORGANIC MOLECULES WITH PROTEINS 101 



that difference is responsible for the behavior. In the case of zinc, we know that 

 for the zinc-dye bond the stabiUty constant is about 0.2 X 10''. This is an as- 

 sociation constant. The zinc-protein bond is some 20 times stronger with a con- 

 stant of 4 X lO''*, so it would seem that in this case the OH would displace the 

 dye and, since there is only a single step here, we will get only one change 

 in the spectrum. 



Now, in contrast, let us look at the copper situation (Fig. 10). Copper tends 

 to form stronger complexes than does zinc with nitrogen atoms and the sta- 

 bility constant for the copper-dye bond in the chelate is a good deal larger than 

 that of zinc. However, the stability constant of the copper for the protein is 

 not of the same order (Fig. 10) and, consequently, the OH ion should tirst 

 displace the metal at P — Cu and, afterward, if you still increase the OH ion 

 concentration, then you will get a split at Cu — D also. 



Thus, the first drop in absorption is due to the break at P — Cu, because when 

 the dye is no longer in the neighborhood of the protein its aqueous environment 

 is different from the protein environment and so its spectrum changes. A second 

 drop occurs when the dye is separated from the copper; its spectrum changes. 

 So we can, I think, account for these differences in behavior in terms of the 

 properties of the metals which are involved and their behavior with respect 

 to the environment. 



Let me turn next to some examples which involve the protein rather than the 

 environment. Again, I would like to give some background information in 

 order to be able to explain some of the new information more clearly. 



We have also examined a number of different dyes (you will notice a great 

 similarity to those which have been used in the hapten work) in binding or in 

 complex formation, in this particular case with serum albumin. Xow, when 

 the dye is bound to serum albumin, its environment is changed, so the spectrum 

 changes (Fig. 11) and this is indicated in the left-hand section; the solid line is 

 the spectrum of the dye, the spectrum of the dye in its protein complex is given 

 by the broken curve. 



For the serum albumins, beta lactoglobulin and casein, which one can complex 

 with these dyes, the spectrum shift is always in the direction shown in the upper 

 left diagram of Fig. 11. The shift is in this direction at any pH for all proteins 

 except one, human serum albumin. Human serum albumin at pH 9 behaves 

 in an anomalous fashion from our reference point. While the spectrum of the 

 dye is not changed, the spectrum of the dye bound to the protein is significantly 

 different, which indicates that the environment of the dye on the protein is dif- 

 ferent at pH 9 than at 6. 



We attempted to localize that change and to interpret it in terms of the spe- 

 cific interaction involved. 



One thing that we knew from early work was that if the charge is removed 

 from the sulfonate substituent (Fig. 11), the ability of this compound to be 



