44 CHARLES TANFORD [3 



as many as ten per cent of its carboxyl, amino, imidazole or guanidine groups 

 anywhere other than in direct contact with solvent. 



Hemoglobin has been cited as a possible exception to this generaliza- 

 tion.'^'^^ This protein undergoes a reaction below pH 4 which is accom- 

 panied by the sudden binding of 18 protons per molecule (molecular weight 

 33,500). The protein acts as if 18 carboxyl groups in their anionic form were 

 not available to titration above pH 4. This conclusion is erroneous, however. 

 The reaction of hemoglobin below pH 4 is accompanied by breakdown 

 of the compact structure (see below). As a result, there is a decrease in 

 electrostatic interactions and this necessarily results in the sudden binding 

 of about 15 protons^^ to sites which were available all the time and were 

 not occupied by protons solely because of the repulsive force of other posi- 

 tively charged sites. Thus, if any of the carboxylate side chains of hemo- 

 globin are not available to titration, the number must be quite small. It is 

 probable that the explanation here given accounts for the apparent unavail- 

 ability of prototropic sites in some other proteins.^ ^ 



Of the side chain groups subject to titration with acid or base, there is 

 one kind which has been found 'unavailable', i.e. presumably in the interior, 

 in some proteins. These groups are the phenohc groups. In ovalbumin^^ 

 all of these groups are unavailable as long as the compact configuration 

 is maintained; in ribonuclease^^ three out of six phenolic groups are un- 

 available; in conalbumin,^"^ too, about half are unavailable. Phenolic groups, 

 however, are not charged at the pH at which these proteins exist in nature. 

 Uncharged phenolic groups have no particular affinity for water and to find 

 some of them in the 'hydrophobic' interior of protein molecules is therefore 

 no contradiction of the generalization made in this section. 



Disulfide bonds. The discussion so far has made only incidental reference 

 to the effect of cross-linkages on the configuration of polymeric chain mole- 

 cules. Most proteins possess such links in the form of disulfide bonds. Their 

 principal effect is to limit the range of possible extended configurations: 

 configurations such as illustrated by Fig. \d may become unattainable as 

 long as disulfide bonds are intact. This factor will indirectly increase the 

 stability of a compact configuration, as compared to that of a randomly- 

 coiled configuration. The chief factor favoring the latter is its high con- 

 figurational entropy. The presence of cross-links, however, decreases the 

 number of possible configurations available to the randomly coiled state, and 

 hence reduces the entropy gain which would result from a transition to this state. 

 There is another possible effect of the presence of disulfide cross-links 

 which works in the opposite direction. To gain maximum benefit from 'hydro- 

 phobic' bonds, electrostatic interactions, etc., requires freedom to bring 

 appropriate portions of a polypeptide chain into each other's vicinity. Di- 

 sulfide bonds interfere with this freedom so that their presence may make 

 a compact structure less stable than it otherwise would have been. 



