INHIBITION BY MACROIONS 457 



bition, showing the importance of the configuration of the polypeptide 

 chains (Tsuyuki et al., 1956). Polyacrylate at concentrations around 0.25- 

 0.5 mg/ml inhibits trypsin at high pH and accelerates catalysis at low pH 

 (Morawetz and Sage, 1955). Denatured hemoglobin, the substrate, has an 

 isoelectric point around 7.8, so that above pH 7.8 the polyacrylate can 

 combine only with the positively charged trypsin (the trypsin-polyacrylate 

 complex has some activity), whereas at lower pH's polyacrylate also binds 

 to the hemoglobin. Since the hemoglobin-polyacrylate complex is more sus- 

 ceptible to trypsin and since hemoglobin is much in excess of trypsin (thus 

 binding most of the polyacrylate), the rate is stimulated at lower pH values. 

 The inhibitions by polyglutamate and polyacrylate are reduced by increas- 

 ing the ionic strength, as expected (Table 1-15-6). It has been shown that 

 copolymers of glutamate with other amino acids (e.g., tyrosine, phenylala- 

 nine, or leucine) are more effective inhibitors than glutamate polymers, 

 but the copolymer of glutamate and alanine is less inhibitory (Rigbi and 

 Sela, 1964). Ornithine polymers or copolymers with ornithine are not inhi- 

 bitory and will reactivate trypsin inhibited by glutamate polymers. This 

 is one instance in which the inhibition produced by glutamate polymers or 

 copolymers is progressive and depends on the incubation time. From the 

 different degrees of inhibition brought about by the various copolymers 

 and the effects of the ionic strength, it was concluded that forces other 

 than electrostatic are involved in the binding. It is interesting that trypsin 

 seems to be particularly susceptible to macroanions, inasmuch as neither 

 chymotrypsin nor papain is inhibited by heparin, although both enzymes 

 are negatively charged at physiological pH. 



The hydrolysis of acetyltyrosine ethyl ester and methylhippurate by chy- 

 motrypsin is inhibited 35-50% by various proteins (seralbumin, ovalbumin, 

 and /?-lactoglobulin ) at concentrations equivalent to the enzyme and in the 

 absence of salt (Hofstee, 1960). Addition of salts, particularly multiply 

 charged ions, reduces or abolishes the inhibition. These proteins are as po- 

 tent inhibitors as the naturally occurring chymotrypsin inhibitors, but differ 

 in not being so sensitive to salt concentration. Carboxymethylcellulose and 

 nucleic acids (both DNA and RNA) also inhibit chymotrypsin (Hofstee, 

 1961). The complexes are dissociated by 100 mM KCl. The inhibition is 

 not complete at maximal binding of nucleic acid, indicating that the active 

 center is not directly involved in the interaction. Methylhippurate was the 

 substrate and if protein substrates had been used it is likely that the active 

 center would not have been accessible. 



Pepsin 



The hydrolysis of hemoglobin by pepsin is rapidly inhibited by poly-L- 

 lysine and this is readily reversible by adding heparin, which complexes 

 with the inhibitor (Katchalski etal., 1954). Cationic polyornithine and poly- 



