H + IONTITRATION AND SA+Ion- ACTIVITY OF TROPOMYOSIN
75
Hydrogen Ion Titration and Sodium Ion Activity of Tropomyosin Solutions by Shozo Iida and Nobuhisa Imai Department of Physics, Faculty of Science, Nagoya University, Chikusa-ku, Nagoya, Japan
(Received June
4, 1968)
Potentiometric titrations and Na+ ion activity measurements in tropomyosin solutions were carried out over a wide range of pH by using an improved hydrogen electrode and a Na+-sensitive glass electrode. The experimental results were analyzed by the theories of rodlike polyelectrolyte, since both tropomyosin monomer and its fibrous polymers, whose transformation is reversibly controlled by salt concentration, are highly charged and rodlike. The titration data indicated the normal titration behavior of the main ionizable groups in high and low salt concentrations. The agreement of the experimental and theoretical values of LinderstrZm-Lang parameter w was satisfactory in acidic and neutral pH’s. A further analysis of tuexptlin the polymer state supported side-overlapping polymerization. The Henderson-Hasselbach relation was useful for the analysis of carboxyl regions, and these plots showed abnormal pH changes in the isoelectric precipitation regions. The small values of tuexptl at high pH were interpreted to be due to the medium penetration into protein resulting from unfolding. The additivity rule for counterion activity was perfectly established at high and neutral pH’s, and a thermodynamic relationship between pH and counterion activity derived from the additivity rule was also experimentally confirmed.
Introduction Tropomyosin, one of the muscle proteins, characteristically polymerizes into fibrous form at low ionic strength and depolymerizes reversibly at high salt concentration.’ Tropomyosin is a rodlike protein with a length2 of 400 A and has a great number of ionizable groups and a high helical content (nearly 100%) at The , ~degree of polymerizaacidic and neutral ~ H ’ s . ~ tion is remarkably affected by pH, showing two maxima at pH 6.5 and S.5,6 which suggests the strong connection of ionizable groups to the polymerization process. Both polymer and monomer states are stable enough for accurate thermodynamic observations. Tropomyosin is thus a very interesting protein for electrochemical investigations as a rodlike polyelectrolyte. It is especially worthwhile to examine whether or not the behaviors of hydrogen and counterions can be explained by the polyelectrolyte theories. I n this study, accurate hydrogen ion titrations are carried out over a wide range of pH in cases of both high and low salt concentrations, and Xa+ ion activity measurements are carefully done at various NaCl concentrations and pH’s. These data are analyzed by the polyelectrolyte theories. I t can be concluded from these results that the behavior of hydrogen ions can be elucidated by these theories and that the ionization constants of the main groups of tropomyosin are not abnormal (i.e.’ no appreciable buried groups are present in tropomyosin). It is also found that the additivity rule for counterions, which is an important rule in usual polyelectrolyte~,~~7 is satisfactorily applicable to the tropomyosin solutions. In our titration experiments, a hydrogen electrode is preferably used to avoid the asymmetry potential which is inevitable in glass electrodes and causes a considerable error in the titrations at high and low pH’s.
Since the usual hydrogen electrodes with platinum black are not available in weakly buffered, neutral solutions because of the error due to the release of acidic substances occluded in t,he electrode, the Hammetttype hydrogen e l e c t r ~ d e , which ~ . ~ is free from. this error, is employed in our experiments. On the basis of our p H measurements, the Hammett electrode is iound to perform well in the titrations of very dilute tropomyosin solutions.
Experimental Section Materials. Tropomyosin was prepared according to the procedure of Bailey1 or Tsao’O with slight modification. After actin was extracted in water from acetone-dried muscle by Mommaerts’ method,” the insoluble residue was washed three times with ethanol and then twice with ether, prior to drying in air overnight. Tropomyosin was extracted a t room temperature from this ether-dried muscle in 1 AI I(C1 over a 10-hr period. Isoelectric precipitate of tropomyosin, made by lowering the pH of the solution to 4.8 with HC1, was gathered and again dispersed in 1 M I
