SynopsisThe objective has been to establish if those ions which are known to change the stability of the struct,ure of proteins, have any influence on the properties of ionizable polypeptides. Potentiometric titrations and complementary optical rotation data are presented for aqueous solutions of poly-L-lysine (PLL) in the presence of KSCN, KC1, and KF, and for poly(L-glutamic acid) (PLGA) in the presence of KSCN, KCl, and LiCI. The following measured quantities which are affected by salt concentration were obtained: intrinsic pK (pKo), slope of pK,,, versus degree of ionization (a) curves, the degree of ionization a t which the helix to coil transition occurs, and the free energy of this transition for the uncharged molecule (AGh",l). The effects of nonspecific salts (KCl arid LiCl for PLL and KSCN and KC1 for PLGA) are small, and about as expected from general e1ectrostat)ic considerations. In line with the observations made with isoelectric and catioriic collagen, specific effects were noted with KSCN-PLL and with LiC1-PLGA. 111 the presence of KSCN, the poly-L-lysine helix becomes stabilized a t much lower degree of ionization than in the presence of KCl, and the slope of the pK,,, versus a plots is greatly reduced. However, AG&l (for the uncharged molecule) is not affected, attd pKo is orily slightly higher. We interpret these data in terms of binding of SCNprimarily to the side-chain aniiiio groups (both to It-NH3+ and to It-NH,). Poly(L-glutamic acid) in LiCl solution has its transit,ion a t the same a value as in KCl solution. However, both the slopes of the pK,,, versus a plots and the absolute values of AGE,, are lower than in KCl solution. We interpret these results ill terms of biiidiiig of Li+ to side chaiiis as well as to the pept,ide bond.

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SynopsisThe effect of salt type and concentration on the t,ransformation 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 a t low salt concentration C,, and decreases a t higher Cs 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, 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, 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. Noneqiiilibrium 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 dihient present in the tendon. Its origin is less clear. For anions such as C1-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 eflects for a given salt soliition makes possible various types of dependence of T, upon Cs: T8 may either continuously decrease or continuously increase with increasing C,, or it may go through a minimum. I n absence of salt,, the cooperative character of the transition at the pH a t which maximum swelling occurs appears extremely reduced. The large swelling maintains the tendon in the elongated state and this simulates a continiioiis decrease of T, on lowering pH. In presence of small quantities of salt, which reduce swelling, the transition is sharp and T, 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, upon Cs is more complex than under isoelectric conditions. There is generally an increase of T, with C, which is equivalent to an increase of the denaturation temperature with C. for helical polyelectrolytes in solution. At higher salt concentrations, however, T. may decrease again, and possibly in...

SynopsisA st.udy of the elastic properties of collagen tendons while in the crystalline state while undergoing shrinkage, and while in the amorphous state was carried out. An additional analysis of x-ray diffraction patterns and birefringence was also performed. Our main observations are the following. The lattice spacings of a quinone-crosslinked collagen tendon are not, altered when the tendon is in equilibrium with KCI or KSCN solutions of different salt concentrations or with acid solutions having different pH. This occurs in spite of large alterations of the modulus of the crystalline tendon in salt and acid solutions. These results indicate that, within the highly ordered framework of the tendon, the salt or acid solution is able to perform a selective disordering action which may involve a considerable fraction of the material before shrinkage takes place. At temperatures and salt concentrations considerably larger than those corresponding to the shrinkage, the tendon exists in a mesomorphic state where residual ordered regions are constrained by the crosslinks in a tactoidal arrangement. The reversibility of length, volume, and birefringence observed within the mesomorphic state attest that it is indeed an equilibrium property of the tendon. Two types of reversible contractile systems can he obtained using the collagen tendon. If full shrinkage is attained, only a fraction of the original length can be recovered on reducing the temperature or salt concentration below the one causing shrinkage. A contraction-extension cycle will then involve the fully denatured and the partly renatured tendon. In order to obtain a reversible contractile system involving recovery of the original length, it is necessary to maintain a more complex "memory" of the original organization than that assured by merely crosslinking in the orientred state and by the occurrence of the mesomorphic phase. This can be done by a selective melting as operated, for instance, by acid solutions. The occurrence of a glasslike state characterized by a high value of the modulus was revealed for the fully amorphous tendon in solutions of salts causing large deswelling. The unperturbed dimensions of collagen molecules are not greatly affected by alteration of salt or salt concentration in the case of KCI and KSCN. The mean-square end-to-end distance r,)* decreases with increasing temperature, i.e., d In rTz j d T = -0.6 X 10-3 deg.-'.Elastic properties of quinone-crosslinked tendons in the amorphous state are similar to those exhibited by other polymer systems crosslinked in the oriented state. Networks formed by chromium salts are, however, peculiar, since they exhibit negative stresstemperature coefficients. The relevance of some of the above results on the aging of connect,ive tissue and on the theories of muscle contraction is pointed out.-

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