Polyelectrolyte properties of sodium hyaluronate. 2. Potentiometric titration of hyaluronic acid

Abstract

Titrations of 0.01 m glucuronic acid (GA) with NaOH were carried out in cells employing glass electrodes with a saturated calomel electrode (cell A) or a silver chloride electrode (cell B). The dissociation constant at zero ionic strength was given for either cell by pK = 3.23 (±0.02), consistent with previous determinations. Corrections for the liquid-junction potential (cell A) led to the expected behavior of apparent pK with concentration of added NaN03. Similar titrations of 0.0085 m hyaluronic acid (HA) from bovine vitreous humor gave essentially linear plots of apparent pK against degree of ionization a over the range a = 0.3-0.8 in the presence of added salt. Least-squares fits to these plots provided slopes which were fitted better as a function of concentration of added salt by the uniformly charged cylinder model than the infinite line charge model of a polyion. The cylinder radius required to obtain a good fit with the structural charge density is about 10 Á, however, which is larger than the structural radius (4-5 Á) of the charge sites. The discrepancy may be due in part to effects of charge discreteness and low dielectric constant of the polyion.The intrinsic dissociation constant for the polymer was estimated to be pK = 2.9 (±0.1), where the large error estimate reflects uncertainties in extrapolation to a = 0. The difference between polymer (HA) and monomer (GA) pK was attributed to effects of substitution at carbon 4 of the monomer. Although data for electrophoretic mobility at zero polymer concentration are limited for hyaluronate, agreement at one ionic strength (0.1 M) of the surface (f) potential calculated from this method and from potentiometric titration suggests that these techniques measure the same potential.

Effects of Polyelectrolyte Chain Stiffness, Charge Mobility, and Charge Sequences on Binding to Proteins and Micelles

“…However, if (pH -pK a ) g 2, we can assume complete ionization, i.e., no charge mobility. This is nearly the case for pectin, for which literature values of pK a vary from 4.0 to 4.4 in pure water, and from 3.5 to 4.0 at I ) 0.05 M. For pectin, pH c values ranged from 5.9 to 4.4, such that the condition (pH -pK a ) g 2 is satisfied at I e 0.1 M. 42 Conflicting values are found for the pK a of HA: Cleland 43 reported 2.8 < pK a < 3.2, depending on I and R; a more recent report gives pK a , in I ) 0.05 M, as 3.5 ( 0.1 independent of R, 44 but these values are also low enough to assume full ionization for HA over most of the pH c range. Therefore, the condition (pH -pK a ) g 2 was satisfied at I e 0.15 M. On the other hand, the larger values of pK a for PAA result in R at pH c ranging from 0.75 to 0.50 as I increases from 0.01 to 0.6 M. Despite this complication of determining the degree of charge for PAA at different condi- tions, we focus in this section on PAA because resolving effects of charge from chain stiffness for HA and pectin is even more problematic.…”

On the difference in ionization properties between planar interfaces and linear polyelectrolytes

“…For linear polyelectrolytes, at intergroup distances somewhere below 1 nm, effects of interactions become observable. This situation is exemplified by polyacrylic acid and hyaluronic acid, where the effects of interactions are weak and titration curves are of the mean-field type, as also revealed by the discrete-charge Ising model (14,30,31).…”

Identification by Integrated Computer Modeling and Light Scattering Studies of an Electrostatic Serum Albumin-Hyaluronic Acid Binding Site

“…Previous studies of synthetic polyelectrolyteprotein systems by such methods, 11,14,24 in addition to fluorescence spectroscopy 29 and electrophoretic light scattering, 30 all reveal a critical pH for complex formation independent of protein or polymer concentration, depending only on ionic strength. Since we find pH c for HA and serum albumin to be always above the pK a of HA, 31 we may consider HA as fully ionized, and therefore interpret changes in pH c in terms of protein charge state alone. The observation of pH c "on the wrong side of the isoelectric point", 11 often seen for protein-synthetic polyelectrolyte systems, suggests the existence of a local protein domain with an effective charge opposite in sign to net protein charge.…”

“…First, to avoid the complicating effects of HA charge variation, the pH was confined to pH > 4.50 (R ) 1). 31 Calculations were then carried out at critical conditions, e.g., pH c ) 5.60, I ) 0.05 M (point a in Figure 4). If a "charge patch" can be correctly identified by computer visualization at point a, that same patch should be seen at another critical point of incipient complexation, e.g., pH c ) 4.70, I ) 0.15 M (point b).…”