INHIBITION BY MACROIONS 459 



Lysozyme 



It is not surprising that this mucolj'lic enzyme is inhibited by a variety 

 of macroanions since its isoelectric point is above 10.5. Heparin is a fairly 

 potent inhibitor of lysozyme (Kaiser, 1953), and it has been stated that 

 this is competitive with substrate (which was a dried preparation of M. 

 lysodeikticus) (Kerby and Eadie, 1953). Inhibition is also exerted by hyalur- 

 onate, polysaccharide of Pneumococcus, polyglutamate, DNA, and RNA 

 (Skarnes and Watson, 1955). Various synthetic polymeric sulfonates also 

 inhibit to different degrees (Heymann et al., 1959). The most potent inhi- 

 bitor yet found for lysozyme is poly glucose sulfate (molecular weight around 

 20,000), although oxidized polyglucose (containing carboxylate groups) 

 is likewise very active; tetraglucose sulfate is without activity (Mora and 

 Young, 1959). These inhibitions are generally reversed by increasing salt 

 concentration or by the addition of a macrocation, such as protamine, to 

 bind the inhibitor. Copolymers of glutamate, tyrosine, phenylalanine, and 

 leucine are potent inhibitors of lysozyme, and the inhibitions can be com- 

 pletely reversed by polylysine (Sela and Steiner, 1963). The greater inhi- 

 bitory activity of the copolymers compared to the homopolymer of gluta- 

 mate is attributed to nonionic bonds; although this is the most likely ex- 

 planation, one must recognize that the charge distribution is quite different 

 in the copolymer relative to the homopolymer. 



Hyaluronidase 



It was noted by Pantlitschko and Kaiser (1951) that hyaluronidase is 

 not significantly inhibited by low molecular weight substances or by high 

 molecular weight substances unless they are esterified with sulfate or are 

 otherwise anionic, and, furthermore, that inhibitory macroanions must be 

 filiform and not globular. Heparin and artificially sulfurated polysaccharides 

 are inhibitory; sulfurated hyaluronate, which can with some justification 

 be thought of as a true analog of hyaluronate, inhibits well. Hyaluronate is 

 a high molecular weight polymer of i\'-acetylhyalobiuronate units and hence 

 contains free C00~ groups; however, sulfuration essentially doubles the neg- 

 ative charge on the molecules, and prevents degradation by the enzyme. 

 Nitrated and acetylated hyaluronates are also inhibitory. A few macro- 

 anionic inhibitors can be mentioned but require no discussion: chitin sul- 

 fates, polymers formed from formaldehyde and various phenolic sulfonates 

 (e.g., hydroquinone, catechol, and resorcinol), polymers formed from form- 

 aldehyde and various hydroxybenzoates, polyesters of phosphate with phe- 

 nols and aniline, polystyrenesulfonate, sulfated pectate, polymethacrylate, 

 amylopectin sulfate, and heparin (Rogers and Spensley, 1954; Bernfeld et 

 al, 1961). 



Alburn and Whitley (1954) suggested that the inhibition of hyaluronidase 



