32 K. LINDERSTR0M-LANG [2 



attitude was taken to the question of the relative velocities of the reactions 



A >-Ai, and Ai >>A2 (see scheme (6)). It was assumed that Aj >'A2 



was the fast reaction so that Eq. (7) assumed the form 



ß=k, (15) 



and it was to ki, the rate constant for the unfolding, that high values of the 

 energies of activation zlH+ and ZlF+ were assigned. The truth probably lies 

 somewhere between the assumption leading to Eq. (8) and to Eq. (15). In 

 the case of PDLA the maximum free energy of unfolding the hehx is that 

 of breaking in the middle, viz., 3000-5000 cal., and AF'^break, the free energy 

 of activation of the breaking, must be at least that high and probably con- 

 siderably higher. From the first term on the right side of Eq. (14) the standard 

 free energy of activation of the hydroxyl ion catalysed exchange may be 

 calculated from the theory of absolute reaction rates. The value obtained, 

 viz. JF!|lea;cA=4700, indicates that somewhere in the alkaline region there 

 is a definite chance that k^ x [QH]^/:i. In the case of insulin, where the 

 breaking of the helices has to involve much greater energies, if our treat- 

 ment used for PDLA applies here, the point where k2[QH]^ki may be 

 reached at lower pH. In agreement with this general picture the overall 

 activation energy for the exchange of PDLA is found to be considerably 

 lower than that found for insulin. ^-^ The portion of the insulin molecule^ 

 in which the ND-groups have half-times of exchange of 24 hours or more 

 at 0° may then be said to have low motility. The easy way of exchange by 

 unwinding the helix is blocked by S-S bonds and the hard one by breaking 

 the hehx is made harder by stabilizing interaction between sidechains.^-^^ 



Native ^-lactoglobulin^*^ in which about 100 hydrogen atoms out of 550 

 do not exchange at all during several days at 38°C at pH 5-4, represents 

 a class of proteins with very low motility, i.e. low ki, but not necessarily 

 low kilk-i. Significant, irreversible denaturation occurs, if at higher tem- 

 peratures (50-60°C) the firmly bound hydrogen atoms are forced to ex- 

 change. Some parts of the ^-lactoglobulin molecule may therefore be said 

 to be kinetically stable but thermodynamically unstable at lower tempera- 

 ture, and a relatively clear distinction between native and denatured forms 

 may be made here. Unfortunately the interesting denaturing effect of high 

 concentrations of urea at 0°^^ could not be studied by back-exchange kine- 

 tics, because ^-lactoglobulin could not be fully deuterated without damage 

 to the protein. Otherwise experiments of this kind would have served to 

 throw considerable light on the process of denaturation. 



It remains now to find an explanation of the fact that PDLA exchanges 

 so relatively slowly with water while the A-chain of insulin and even oxi- 

 dized ribonuclease react almost instantaneously. As pointed out by Schell- 

 man^^ and by Harrington and Schellman,^^ the tertiary structure plays a 

 very important role in the stabilization of hehces, which when 'naked' seem 

 to be very loose with a. AFr close to zero. Since PDLA is unassociated in 



