Interaction between proteins and salt solutions. III. Effect of salt type and concentration on the shrinkage temperature

The effect of salt type and concentration on the transformation of an oriented crystalline collagen tendon into a crosslinked network under conditions of equilibrium swelling was investigated. Our main observations are the following. The degree of swelling of crystalline tendons increases at low salt concentration C s , and decreases at higher C s for a wide variety of salts. The observation is not reconcilable with swelling taking place in interfibrillar spaces or structural voids. Within the tropocollagen units and at their ends, regions of reduced organization are postulated (as suggested by Bear and by Schmitt) which are able to interact with the diluent in the amorphous‐like manner. At least four different factors should be considered in assessing the role of salt and salt concentration on the shrinkage temperature T s under isoelectric conditions. They are: ( 1 ) specific effects, ( 2 ) diluent effects, ( 3 ) crosslinking effects, and ( 4 ) nonequilibrium effects. The diluent effects are correlated with the salting‐in–salting‐out power of the ions which was characterized in Part I of this series. Smaller amounts of diluents are generally available to the tendon when the salt has a higher salting‐out power, and this corresponds to higher shrinkage temperatures, other conditions being the same. The crosslinking effect raises T s due to a reduction of the diluent content and, probably for p ‐benzoquinone and formaldehyde, also to a reduction of the conformational entropy in the molten state. Nonequilibrium effects arise from the fact that shrinkage and recrystallization are kinetically hindered when the tendon is highly deswollen in strong salting‐out solutions, or when the salt has a crosslinking power. The specific effect is the only effect which is not related to the amount of diluent present in the tendon. Its origin is less clear. For anions such as Cl − and SCN − , it is possibly related to an ability of the ion to prevent intersegmental hydrogen bonding and water carbonyl bridges. The competition of several of the above effects for a given salt solution makes possible various types of dependence of T s upon C s : T s may either continuously decrease or continuously increase with increasing C s , or it may go through a minimum. In absence of salt, the cooperative character of the transition at the pH at which maximum swelling occurs appears extremely reduced. The large swelling maintains the tendon in the elongated state and this simulates a continuous decrease of T s on lowering pH. In presence of small quantities of salt, which reduce swelling, the transition is sharp and T s is decreased with pH up to pH 2, when maximum swelling occurs, and then reincreases on further lowering of the pH. The dependence of T s upon C s is more complex than under isoelectric conditions. There is generally an increase of T s with C s which is equivalent to an increase of the denaturation temperature with C s for helical polyelectrolytes in solution. At higher salt concentrations, however, T s may decrease again, and possibly increase again at still higher salt concentrations, depending upon the effect of the salt solution in the isoelectric zone.