[Special role of phosphate in the stability of the protein to the destruction by polyelectrolyte].

X-ray analysis shows the presence of specific anion-binding sites in proteins for sulfate, citrate and phosphate ions, but the functional role of these anions is not always clear. Thus, it is unknown which of the two types, mono- or divalent phosphate, plays an important role in the stability of proteins to stress effects on cells. In the present work, the influence of phosphate, sulfate, and chloride salt on the stability of lactate dehydrogenase (LDH) to its destruction by poly(styrenesulphonate) (PSS) was investigated by the methods of steady-state kinetics and the own protein fluorescence. The analysis was based on the differences between the influence of phosphate and sulfate ions on the process at two pH values, 6.2 and 7.0, at which the ratio of mono- and divalent phosphate changed, whereas sulfate remained in the divalent form. It was shown that the difference between the influence of phosphate and sulfate ions increased with increasing pH, which indicates that divalent phosphate ions much more effectively stabilized LDH compared to sulfate and monovalent phosphate. The differences in the effect of sulfate and chloride salts on the protein corresponded to differences in ionic strength of their solutions. The study of the own fluorescence of LDH in the complex with PSS showed that the rate of fluorescence quenching and the amplitude of the fast stage significantly decreased with increasing concentration of divalent phosphate in solution, as compared to the same effect in the presence of sulfate anions. The conclusion was made that, from two anion-binding sites in the LDH molecule, the intersubunit center is most important in stabilizing the protein to the destruction by polyelectrolyte, and from two phosphate anions, hydrophosphate HPO4(-2) plays the stabilizing role.

[Interaction of protein with charged colloidal particles].

The functional state of three proteins of different molecular weight (urease, lactate dehydrogenase, and hemoglobin) in the presence of the linear polyelectrolytes poly(allylamine hydrochloride) (PAA) and sodium poly(styrenesulfonate) (PSS) in the dissolved state and of the same polyelectrolytes bound to the surface of microspheres has been investigated. Microspheres were prepared by consecutive absorption of oppositely charged polyelectrolytes so that the outer layer of the shell was PAA for the acidic protein urease, and PSS for the alkaline proteins LDH and hemoglobin. It was shown that the dissolved polyelectrolyte completely inactivates all three proteins within one minute with a slight difference in the time constant. (By Hb inactivation are conventionally meant changes in the heme environment observed from the spectrum in the Soret band.) In the presence of microspheres, the proteins were adsorbed on their surface; in this case, more than 95% of the activity was retained within two hours. The proportion of the protein adsorbed on microspheres accounted for about 98% for urease, 72% for Hb, and 35% for LDH, as determined from the tryptophan fluorescence data. The interaction of hemoglobin with another type of charged colloidal particles, phospholipid vesicles, leads to the destruction of the tertiary structure of the protein, which made itself evident in the optical absorption spectra in the Soret band, as well as the spectra of tryptophan fluorescence and circular dichroism. In this case, according to circular dichroism, the percentage of alpha-helical structure of Hb was maintained. The differences in the physical and chemical mechanisms of interaction of proteins with these two types of charged colloidal particles that leads to differences in the degree of denaturing effects are discussed.

[Thermal stability of lactate dehydrogenase in the complex with the anion polyelectrolyte poly(styrene sulfonate)].

The temperature stability of the cytoplasmic enzyme of the glycolysis of lactate dehydrogenase from a pig muscle (isoenzyme M4) in a complex with the anion polyelectrolyte poly(styrenesulfonate) has been investigated by the methods of adiabatic differential scanning microcalorimetry, the own protein fluorescence, and circular dichroism. Calorimetric investigations of complex of lactate dehydrogenase with poly(styrenesulphonate) in 50 mM phosphate buffer at pH 7.0 have shown that the temperature of the transition and enthalpy of lactate dehydrogenase thermal denaturation sharply decreases with growing weight ratio poly(styrenesulphonate)/lactate dehydrogenase, though at 20 degrees C the enzyme activity of lactate dehydrogenase remains unchanged for several hours irrespective of the addition of poly(styrenesulphonate). The addition of phosphate ions to the solution enhances the resistance of lactate dehydrogenase to both thermal denaturation and inactivation by polyelectrolyte. The data obtained are interpreted from the viewpoint of a special role of two anion-binding centers in intersubunits contacts of lactate dehydrogenase, which enhance its resistance to both thermal denaturation and destruction by polyelectrolyte.