456 2, ANALOGS or enzyme reaction components 



zation state of groups with which the inhibitor reacts, and in all cases in 

 which this has been studied a marked dependence on the pH has been 

 demonstrated. An increase in the ionic strength should reduce such inhibi- 

 tions because of the competition of the small ions for the enzyme and ma- 

 croion groups, and this has been repeatedly confirmed experimentally (data 

 for the inhibition of trypsin by polyglutamate are given in Table 1-15-6). 

 Hydration of the ionic groups must also be a significant factor in reducing 

 the inhibitions from what might be expected on the basis of interactions 

 in a vacuum, so that anything which modified the extent of hydration of 

 either enzyme or macroion might secondarily affect the inhibition. A final 

 factor which can markedly reduce such inhibitions is the presence of ma- 

 croionic impurities in the preparation if one is not working with pure en- 

 zymes. It has been shown many times that inhibitions by macroions can 

 be prevented or actually reversed by other macroions of opposite charge to 

 the inhibitor. Such results are not particularly significant since they imply 

 only that the inhibitor can also bind to nonenzyme macroions, a fact which 

 can be better demonstrated with other techniques, but they emphasize the 

 possible importance of such impurities in the studies on macroionic inhi- 

 bitions. 



Trypsin and Chymotrypsin 



The isoelectric point of trypsin is close to pH 11 and that of casein is 

 between 4 and 4.5; thus the hydrolysis of casein involves the interaction 

 of a macrocationic enzyme with a macroanionic substrate at pH values near 

 neutrality. Since heparin, a strongly negatively charged sulfated polysac- 

 charide, was known to form complexes with positively charged proteins, 

 Horwitt (1940) examined its action on trypsin and found a rather potent 

 inhibition at pH 7.3. Inhibition does not occur unless the enzyme is incubat- 

 ed with heparin before the addition of the casein, possibly indicating a 

 competitive type of interaction. Acidification to pH 3 leads to a dissociation 

 of the trypsin-heparin complex with restoration of full activity. The pH^pt 

 for trypsin is possibly shifted from 8.4 to lower values by heparin (Glazko 

 and Ferguson, 1940); it is not known if this means that enzyme combined 

 with heparin can act on casein — it is difficult enough to understand the 

 pHopt of proteolytic enzymes in the absence of inhibitors. The distance 

 between sulfate groups in heparin is 10.2 A, which is approximately equiv- 

 alent to 3 peptide residues in proteins, so it was suggested by Kornguth 

 and Stahmann (1960) that heparin may bridge the active site by combining 

 with cationic groups on either side. The active site appears to be covered, 

 since the hydrolysis of benzoylarginamide by trypsin is inhibited. Poly-or- 

 L-glutamate and polycysteate also inhibit trypsin, but poly-y-D-glutamate 

 does not, and this is probably correlated with the different distances between 

 C00~ groups in these macroanions. Poly-D-lysine inhibits the tryptic hy- 

 drolysis of poly-L-lysine, equimolar concentrations giving complete inhi- 



