DENATURATION 255 



in kinetic studies. Stearn and co-workers (728,2016) interpret this as the 

 reflection of the considerable entropy change which accompanies denatura- 

 tion, the almost unique configuration in the native protein being displaced 

 by a more disordered structure. Other workers, such as Steinhardt (2020) 

 La Mer {1636), and Neurath and co-workers [20^6) contest this interpre- 

 tation, and suggest that, if proper allowance is made for the effect of ;;H, the 

 energy of activation is found to be much smaller. The calculation of the 

 actual number of bonds broken depends on the energy of activation assumed 

 to be required for the rupture of a hydrogen bond or a salt linkage (cf. I^61, 

 p. 4'-27; 20J^6, p. 204). Neurath concludes that denaturation by acid in the 

 /)H range 4.1 to 4.6 follows the breaking of only two essential hydrogen bon4s. 



While further work will doubtlessly establish accepted values for the 

 energies of activation, this approach does not appear able to indicate whether 

 the linkages broken on denaturation are hydrogen bonds or salt linkages. 

 Mirsky and Pauling {196U) have given a qualitative explanation of some of 

 the features of denaturation which, while not excluding the possible role of 

 salt linkages, makes it appear likely that hydrogen bonds are involved. The 

 action of acids and alkalis is explained by their ability to rupture the hydrogen 

 bridge by supplying or removing protons from the bond; while urea, alcohol, 

 and salicylate denature because of their ability to form hydrogen bonds 

 themselves with one of the partners of the linkage, thus displacing the other. 

 The eflfect of pH on the sensitivity of proteins to other reagents is interpreted 

 as the rupture of a few of the hydrogen bonds with consequent loosening of 

 the protein structure, so that the entry of the larger molecules is facilitated 

 {cf. 212k, p. 146). 



On denaturation the isoelectic point of hemoglobin and globin is shifted 

 by about one pH unit toward higher values. Laporta (16^9) showed by 

 electrometric titrations on native and denatured ox globin that s. dissociation 

 with a pK value of 8.2 in the native protein was shifted to a pK value of 

 10.2 on denaturation. 



4.3.3. Reversible Denaturation 



4.3.3.1. Action of Alkali. Provided the ionic strength is low, the denatu- 

 ration is probably proportional to the hydroxyl ion concentration {1162), 

 although this would merit reinvestigation in the light of present theories of 

 denaturation. Increase of the ionic strength above a certain optimum retards 

 the rate of denaturation at a given pYL {1727). Oxyhemoglobin is denatured 

 by alkali more slowly than is hemi'globin. Differences are present both 

 between the rates at which hemoglobins from adults of different species are 

 denatured under identical conditions, as well as between the rates of denatu- 

 ration of fetal and adult hemoglobin (see Chapter \TI, Section 6.1.2. and 

 6.2.2.). 



Roche and Chouaiech {2305,2310,2312) found that hem/chrome formed 

 by denaturing horse or ox hemoglobin at pH 11.6 to 11.8 could be renatured 

 by dialysis at 0° C. They were able to renature 70 to 80% of the original 

 pigment. 



Hill {1275) has observed that in certain conditions the effect of alkali may 



