96 I. M. KLOTZ 



However, when zinc is put in, it is possible to form a chelate of the type shown. 

 It is also known that the zinc can be bound to the protein so that it seems likely 

 that the zinc actually forms a bridge. However, that is not an absolutely cer- 

 tain interpretation, at least with the information I have given you so far, be- 

 cause it is also known that if you add hydrogen ions to a solution you can 

 increase the extent of binding of a large number of dyes and I do not think 

 anyone would be foolish enough to say that the hydrogen ion acts as a tetrafur- 

 cated hydrogen bond. What hydrogen ion does, as is well known, is to cause 

 some change in the protein; change in charge, change in its structure, some- 

 times change in its degree of folding. So, of course, it is possible that the zinc 

 operates by a similar mechanism; that is, that it too causes some change in the 

 protein rather than acting as a bridge to the organic molecule. 



One of the experiments which we think rules this out is essentially the fol- 

 lowing. If zinc had its effect on the binding of this dye, due to some change in 

 the protein, then if we took a structurally analogous dye in which the pyridine 

 ritrogen is at the extreme left of the structure (Fig. 4) so that the zinc cannot 

 form a chelate, we should still obtain the same effect in the presence of zinc. 

 You have a molecule exactly the same size, and with the same type of basic 

 groups involved. If the effect of the zinc is on the protein, then zinc ought to 

 increase the binding of the parapyridine dye just as much as it increases that 

 of the ortho one shown in Fig. 4. As a matter of fact, the parapyridine dye acts 

 in the presence of zinc just as if zinc were not present. It is bound to the same 

 extent in the absence of zinc as the ortho (Fig. 4) is bound, but if zinc is put in, 

 the ortho is bound much more strongly and the para dye is not bound to any 

 extent above that to which it would be bound if there were no zinc present. 



So I think that this experiment indicates quite clearly that it is essential 

 that the zinc be able to form a chelate with the dye. Consequently, since we 

 know by separate experiments that the zinc is bound to the protein — in fact, 

 I shall show you some of those results, too, in passing — it is quite clear that the 

 zinc is acting as a bridge in the ternary complex (Fig. 4). 



Let us look at this matter in a little more detail. It takes a relatively large 

 amount of routine manipulation to get the extensive data of binding as a func- 

 tion of concentration of the type shown in Fig. 3. Instead of that, because we use 

 dyes, we can do the following. When the dye is bound to the protein through 

 zinc or otherwise, it changes its absorption so that at least semiquantitative^ all 

 we need to do is follow the increase in absorption at a suitably chosen wave 

 length to have a measure of extent of binding of dye in the presence of zinc. 



I can summarize the data (Hughes and Klotz, 1956) in terms of an optical 

 density experiment of the type shown in Fig. 5. The greater the optical density, 

 the more the binding, and I have summarized the data for a fairly extensive 

 pH range, out to about 11. You can see that first there is an increase in the ex- 

 tent of binding as we increase the pH from approximately 6 to 7.5, and there- 

 after there is a drop in the extent of binding. 



