788 7. MERCURIALS 



this have been discussed in the previous section and we shall now inquire 

 what further evidence on this important problem has come to light. One 

 of the more obvious reasons for suspecting denaturation is the progressive 

 development of irreversibility during contact with the mercurial, such as 

 has been reported for cholinesterase (Goldstein and Doherty, 1951), pros- 

 tatic phosphomonoesterase (Tsuboi and Hudson, 1955 a), muscle aldolase 

 (Swenson and Boyer, 1957). and muscle 3-PGDH (Elodi, 1960) — to men- 

 tion only a few instances, in most cases reversal being attempted with glu- 

 tathione or dimercaprol. Of course, one might attribute failure to reverse 

 to very tight binding to the enzyme, but the progressive increase in the 

 irreversibility points more to structural changes. The question often remains 

 as to whether these changes are responsible for the inhibition or are super- 

 imposed upon it, i.e., inhibition followed by inactivation. 



Elodi (1960) investigated the changes in several properties of pig muscle 

 3-PGDH treated with p-MB, and found significant deviations in the op- 

 tical rotation and the intrinsic viscosity, the latter increasing linearly with 

 the equivalents of mercurial added. The following phases were postulated: 

 (1) an initial reversible binding and inhibition, (2) a progressive disintegra- 

 tion of the secondary structure of the enzyme as a result of the blocking of 

 SH groups, this probably involving an unfolding of the polypeptide helices, 

 and (3) polymerization and precipitation consequent to the freeing of 

 groups which form intermolecular bridges. The simultaneous changes in the 

 activity, NADH binding, and rotatory dispersion of yeast alcohol dehydro- 

 genase treated with p-MB led Wallenfels and Miiller-Hill (1964) to postulate 

 that modifications of the secondary and tertiary protein structure occur 

 when the SH groups are blocked. Reaction of 10 SH groups on muscle 

 aldolase with p-MB does not reduce the activity but the susceptibility to 

 tryptic digestion is increased (Szabolcsi and Biszku, 1961). Untreated al- 

 dolase or treated enzyme in the presence of fructose-diP is not digested by 

 trypsin; thus the substrate apparently protects the active center, and per- 

 haps the entire molecule, from hydrolysis. It is thought that reaction of the 

 first 7 free SH groups labilizes the tertiary structure of the enzyme, and 

 from then on a progressive denaturation occurs. Addition of substrate may 

 restore to some degree the normal structure. The inhibition that occurs 

 later or with excess p-MB does not seem to be directly related to mercaptide 

 formation but dependent on the structural changes when they have pro- 

 ceeded past a certain point. Another interesting approach was made by 

 Massey (1958) in showing that the chelation of the nonheme iron of suc- 

 cinate dehydrogenase by o-phenanthroline is accelerated by treatment with 

 p-MPS, this being interpreted as a structural change exposing the iron. 

 The SH groups of yeast hexokinase can be titrated with p-MB in the pre- 

 sence of glucose without loss of activity, but spontaneous denaturation 

 quickly follows (Fasella and Hammes, 1963). Glucose-6-P does not prevent 



