ALBERT

Notes: The action of dilute aqueous mineral acids on α,α-diphenyl-β-picryl hydrazyl (DPPH) has been studied and shown to result in the rapid loss of the characteristic color of the radical. Hydrobromic and hydrochloric acids yield α,α-diphenyl-β-picryl hydrazine (DPPH-H) and the corresponding halogen-substituted α,α-diphenyl-β-picryl hydrazine. Hydroiodic acid gives DPPH-H, iodine, and a small quantity of DPPH. A number of organic acids cause similar reactions. Kinetic studies on the hydrobromic acid system indicate that the reaction is first-order with respect to: hydrogen ion concentration, DPPH concentration, and anion concentration. The ability of the anion to undergo one-electron transfer and to dimerize are also important, and the strongest nucleophile gives the fastest reaction. A mechanism which involves ionic attack is proposed to account for the reaction of DPPH with acid. The limitations of DPPH in studying reaction mechanisms are then discussed in relation to the proposed ionic mechanism.

Notes: The synthesis and polymerization reactivity of N[2-(2-methyl-4-oxopentyl)]-acrylamide (diacetone acrylamide) is described. This reactive vinyl monomer is prepared in good yield from acetone, acrylonitrile and sulfuric acid. Diacetone acrylamide is a white crystalline solid with the water solubility characteristics of acrylamide and the organic solvent solubility of N-alkyl acrylamides. Diacetone acrylamide readily homopolymerizes and forms copolymers with a wide variety of comonomers. Reactivity ratios in the copolymerization of diacetone acrylamide with styrene and methyl methacrylate are reported and the Alfrey-Price Q and e values calculated. These results, along with data from the copolymerization of diacetone acrylamide with a group of typical vinyl monomers, are presented to indicate the high reactivity of this new monomer.

Notes: Calorimetric measurements of the heat of the addition of the second strand of poly U to poly (A + 2U) to form the three-stranded poly (A + 2U) complex in 0.1M and 0.5M NaCl at 24 and 37°C. are reported. A value of ΔH = -3800 cal./mole of poly (A + 2U) formed was found to be fairly insensitive to the experimental conditions employed. The heat of the addition of the third strand to the preformed poly (A + U) helix is considerably less exothermic than the heat of reaction between poly A and poly U to form poly (A + U). The insensitivity of the heat of addition of the third strand (poly U) to changes in salt concentration and temperature lends qualitative support to the earlier hypothesis that the major portion of the variation of the ΔH of the poly A and poly U reaction with experimental conditions arises from differences in the conformation of poly A. Combining the information obtained in this study for the ΔH of the reaction forming poly (A + 2U) with data for the ΔH of the formation of poly (A + U) indicates that the conversion of poly (A + U) to poly (A + 2U) is opposed by an enthalpy change which increases with temperature. Extrapolation of these values to 52°C. where poly (A + U) is transformed to poly (A + 2U) in 0.5M NaCl leads to a value of ΔH = +3800 cal./mole (A + 2U) formed and ΔS = 11.5 cal./mole of (A + 2U) formed. It is concluded from the calorimetric data that the driving force for the poly (A + U) to poly (A + 2U) conversion reaction is the favorable entropy change.

Notes: 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 Cs, and decreases at 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 Ts 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 Ts 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 Ts upon Cs: Ts may either continuously decrease or continuously increase with increasing Cs, 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 Ts on lowering pH. In presence of small quantities of salt, which reduce swelling, the transition is sharp and Ts is decreased with pH up to pH 2, when maximum swelling occurs, and then reincreases on further lowering of the pH. The dependence of Ts upon Cs is more complex than under isoelectric conditions. There is generally an increase of Ts with Cs which is equivalent to an increase of the denaturation temperature with Cs for helical polyelectrolytes in solution. At higher salt concentrations, however, Ts may decrease again, and possibly increase again at still higher salt concentrations, depending upon the effect of the salt solution in the isoelectric zone.

Notes: Intrinsic viscosity-temperature studies for isoelectric gelatin in KCl and KSCN aqueous solutions and equilibrium degree of swelling, ν-1, measurements for amorphous crosslinked rat tail tendons in the same solutions were carried out. On increasing salt concentration Cs, both [η] and ν-1 increase, go through a maximum, and then decrease at high Cs, KCl being more effective than KSCN in bringing about this decrease. The trend observed is similar to the variation of solubility of polypeptides and soluble proteins with Cs. By regarding a water-salt solution of a given Cs as a single diluent interacting with the protein modified by solvation and binding of ions, usual polymer solution theories were applied to the experimental results. Thus, quantities related to the entropy and enthalpy components of the excess chemical potential of the diluents were obtained. The data indicate that the initial increase of [η] and ν-1 with Cs results from the balance of an enthalpy component which, on increasing Cs, becomes less favorable to dilution and an entropy component which, conversely, becomes more favorable. The subsequent decrease of [η] and ν-1 with Cs is due to the prevailing of the enthalpy component. The maximum is reached at higher Cs for KSCN than for KCl, primarily because of the large increase in the entropy component operated by the former salt. The increase of the entropy parameter with Cs is associated to a breaking down of the coordinated water structure and to an alteration of the conformation of the macro molecules due to ion-dipole interaction and to ion absorption. The decrease in exothermicity of dilution with Cs indicates a reduced thermodynamic affinity of the diluent toward the polymer which probably results from alteration of the nature of both polymer and diluent.

Notes: Aqueous solutions of ribonuclease and human serum albumin were subjected to periods of controlled heating and the nuclear magnetic spin-lattice relaxation times, T1, of the water protons measured. The heat treatment causes an initial increase in the relaxation times of the water protons. These T1 variations indicate that a configurational change, compatible with a disordering of the protein structure occurs, which involves an increase in various modes of internal mobility. The T1 measurements also indicate a close, ordered association of the water molecules and the protein.

Notes: The melting characteristics of unfractionated yeast s-RNA in neutral aqueous solution have been examined by high-resolution proton magnetic resonance spectroscopy. These studies indicate that the bases of s-RNA become rotationally activated at lower temperatures than their associated ribose units. These observations, as well as the recently determined base sequence of an alanine-specific s-RNA, suggest that base complementarity may not be the primary determinant in the stabilization of the secondary structure of s-RNA. In addition, evidence for exchange of C8-protons of purine bases of s-RNA with D2O was obtained at temperatures above 70°C.