INHIBITION BY MACROIONS 455 



extensively but one can imagine some interesting phenomena in this con- 

 nection. The initial binding of the macroion to the enzyme would not be 

 expected to be that for maximal interaction because the first contact be- 

 tween them would be random. Inasmuch as most macroionic inhibitions are 

 readily reversible, it is likely that the polymer would move on the enzyme 

 surface until near maximal or maximal interaction occurs. The inhibition 

 for this reason might increase with time, the rate depending on several 

 factors, such as the binding affinity of the individual group interactions 

 and the flexibility of the polymer, and progressive developments of inhi- 

 bition have been experimentally observed. Inasmuch as many configura- 

 tions of the macroion on the enzyme surface may be characterized by inter- 

 action energies very near the maximal, it is likely that even at equilibrium 

 each enzyme molecule will not be inhibited to the same degree, especially 

 for those enzymes acting on small substrates, since the inhibition will usual- 

 ly be due to a steric interference by a polymer chain passing over or near 

 an active site. 



Factors Determining the Degree of Inhibition 



The most important factors relating to the inhibitory macroion would be 

 (1) the molecular weight or polymer length, (2) the density of ionic groups 

 on the polymer, or the repeat distance between them, (3) the over-all con- 

 figuration of the polymer, i.e., linear, branched, or globular, and (4) the 

 flexibility of the polymer. The last factor is perhaps very important but 

 has been generally ignored. If one assumes an approximately globular en- 

 zyme, the net binding energy and the degree of inhibition may well depend 

 on the ability of the polymer to conform to the enzyme surface, specifically 

 to wrap around it so that interactions between many ionic groups can take 

 place. Some of the ionic polysaccharides must not be too flexible and this 

 may limit the effects they have on certain enzjTnes, while the synthetic 

 macroions vary in flexibility over a wide range. Entropy factors must be 

 very significant in the binding of macroions, and would to a great extent 

 depend on the deviation of the bound polymer from its statistical configu- 

 ration in solution. A polysaccharide or polypeptide macroion of molecular 

 weight 10,000 would contain roughly 35 units and the total extended length 

 would be 200-250 A if linear. Such a macroion might be able to encircle 

 an average enzyme 1 or 2 times or, if it is randomly distributed over the 

 enzyme surface, would cover very roughly about 10-15% of the enzyme. 

 Many macroions used to inhibit enzymes are, of course, much larger, often 

 being of molecular weights of 100,000 or over. 



Two characteristics of the media used in inhibition studies are particu- 

 larly important, namely, the pH and the ionic strength, since the interac- 

 tions between enzyme and macroion are mainly of the ion- ion type. The 

 pH will determine the net charge on the enzyme and occasionally the ioni- 



