Self-association of alpha-chymotrypsin at low ionic strength in the vicinity of its pH optimum.

The self-association of alpha-chymotrypsin and its di-isopropyl phosphoryl derivative in in I0.03 sodium phophate buffer, pH7,9, was investigated by velocity sedimentation, equilibrium sedimentation and difference gel chromatography. No differences between the native and chemically modified enzyme were observed in the ultracentrifuge studies, and only a marginal (0.6%) difference in weight-average elution volume was detected by difference gel chromatography of 5g/litre solutions on Sephadex G-75. From quantitative analyses of sedimentation velocity and sedimentation-equilibrium distributions obtained with iPr2P (di-isopropylphosphoryl)-chymotrypsin, the polymerizing system is postulated to involve an indefinite association of dimer (with an isodesmic association constant of 0.68 litre/g) that is formed by a discrete dimerization step with equilibrium constant 0.25 litre/g. In addition to providing the best fit of the experimental results, this model of chymotrypsin polymerization at low ionic strength is also consistent with an earlier observation that dimer formation is a symmetrical head-to-head phenomenon under conditions of higher ionic strength (I0.29, pH7.9) where association is restricted to a monomer-dimer equilibrium. It is proposed that the dimerization process is essentially unchanged by variation in ionic strength at pH7.9, and that higher polymers are formed by an entirely different mechanism involving largely electrostatic interactions between dimeric species.     

The effect of alkaline earth cations and of ionic strength on the dissociation of earthworm hemoglobin at alkaline pH

1. The effect of alkaline earth cations on the dissociation of the extracellular hemoglobin of Lumbricus terrestris and the effect of ionic strength on the dissociation of the hemoglobins of L. terrestris and Tubifex tubifex at concentrations of ca 2.5 mg/ml, over the pH range 9.0-10.5 was investigated using ultracentrifugation to separate the dissociated from the undissociated molecules. 2. Mg(II), Ca(II) and Sr(II) at concentrations of up to 0.2 M, decreased the dissociation of Lumbricus oxyhemoglobin from 70% at pH 9.0 and 100% at pH 9.5 and higher, to 20-30% at 0.05 M. The three cations were equally effective in decreasing the extent of dissociation of L. terrestris oxyhemoglobin over the pH range 9.0-10.5, with a K1/2 of ca 10 mM. 3. The dissociation of L. terrestris oxyhemoglobin over the pH range 9.0-10.5 was decreased only to 50-60% in the presence of up to 0.5 M NaCl or KCl; there was no further decrease in dissociation at concentrations of the two salts up to 1.5 M. 4. The dissociation of T. tubifex oxyhemoglobin over the pH range 9.0-10.0 was decreased from 100% to ca 40-50% in the presence of 0.5 M NaCl or KCl with little or no change at higher concentrations. At pH 10.5 and 11.0 the decrease in dissociation was more gradual, reaching ca 50% at 1.5 M NaCl.     

Arsenazo III-Ca2+. Effect of pH, ionic strength, and arsenazo III concentration on equilibrium binding evaluated with Ca2+ ion-sensitive electrodes and absorbance measurements.

Equilibrium binding properties of the metallochromic indicator Arsenazo III (AIII) were characterized by Ca2+ and acid/base titration. Free calcium was measured directly with Ca2+ ion-sensitive electrodes. Absorbance changes were measured by both a conventional scanning spectrophotometer and a dual wavelength spectrophotometer. Acid/base titration of AIII in conjunction with Ca2+ ion-sensitive electrode measurement and absorbance changes indicate that pH can change AIII absorbance through a change of the K(D) for Ca-AIII formation, and, in addition, that there is a pH-specific component that is not dependent on Ca-AIII formation. THe dissociation constant (K(D)) of AIII varied not only with pH, but with ionic strength and AIII concentration. Studies conducted to examine AIII-Ca stoichiometry resulted in different initial conclusions, depending on the method of analysis. Log delta A-log AIII relations were in accord with previously published results, which indicate that more than one AII binds to one Ca2+ ion. But Job plots. Scatchard analysis, and Hill plots all indicated 1:1 binding. THe method of absorbance measurement, i.e., scanning or dual wavelength did not influence the results. these findings were reconciled on the basis of changes in K(D) with AIII concentration and ionic strength. In 200 mM KCl, K(D) of AIII-Ca varies by a factor of 7 between 10(-4.3) and 10(-3) M AIII. Thus, a disproportionately large amount of Ca-AIII is formed as AIII concentration is increased, which results in slopes greater than unity for log delta A-log AIII relations.     

Nonideal size-exclusion chromatography of proteins: Effects of pH at low ionic strength

Ideal size-exclusion chromatography separates molecules primarily on the basis of hydrodynamic volume. This is achieved only when the chromatographic support is neutral and the polarity nearly equal to that of the mobile phase. When this is not the case, the support surface may begin to play a role in the separation process. As the magnitude of surface contributions becomes larger, the deviation from the ideal increases. Because the separation mechanism is different than that of ideal size-exclusion chromatography, selectivity could be increased in nonideal size-exclusion chromatography. This paper explores the use of size-exclusion chromatography columns with mobile phases that cause proteins to exhibit slight deviations from the ideal size-exclusion mechanism. Although there are many ways to initiate nonideal size-exclusion behavior, the specific variable examined in this study is the influence of pH at low ionic strength. Individual proteins were chromatographed on SynChrom GPC-100, TSK-G2000SW, and TSK-G3000SW columns at low ionic strength. It was found that a protein could be selectively adsorbed, ion excluded, or chromatographed in an ideal size-exclusion mode by varying mobile-phase pH relative to the isoelectric point of the protein. In extreme cases, molecules could be induced either to elute in the void volume or beyond the volume of total permeation. It is postulated that these effects are the result of electrostatic interactions between proteins and surface silanols on the support surface. Optimization of size-exclusion separations relative to protein isoelectric points is discussed.     

Reversible and non-reversible thermal denaturation of lysozyme with varying pH at low ionic strength

DSC analysis has been used to quantify the reversibility of unfolding following thermal denaturation of lysozyme. Since the temperature at which protein unfolding occurs, T_m, varies with different solution conditions, the effect on the melting temperature and the degree of refolding after thermal denaturation in low ionic strength sodium phosphate buffers (5–1000 mM) over a range of pH (5–9) in the presence/absence of disaccharides is examined. This study compares the enthalpies of unfolding during successive heating cycles to quantify reversibility following thermal denaturation. The disaccharides, trehalose and maltose were used to assess if the disaccharide induced increase in T_m is reflected in the reversibility of thermally induced denaturation. There was extensive overlap between the T_m values where non-reversible and reversible thermal denaturation occurred. Indeed, for pH 6, at the highest and lowest T_m, no refolding was observed whereas refolding was observed for intermediate values, but with similar T_m values having different proportions of refolded protein. We established a method to measure the degree of reversible unfolding following thermal denaturation and hence indirectly, the degree to which protein is lost to irreversible aggregation, and show that solution conditions which increase melt transition temperatures do not automatically confer an increase in reversibility. This type of analysis may prove useful in assessing the stability of proteins in both the biopharmaceutical and food industries.     

Analysis of deoxyribonuclease I and II activity as a function of ionic strength and pH]

The action of deoxyribonucleases I and II has been studied as a function of ionic strength and pH, in the light of the theory of the ionic control of biochemical reactions (P. Douzou and P. Maurel (1977) Proc. Nat. Acad. Sci. USA, 74, 1013-1015). The pattern of DNA degradation by the two enzymes fits the general principles of the theory. However, the activity of DNAase II, a dimeric, basic protein (pI = 10,2) appears to be scarcely modulated by variables such as ionic strength and pH. This is reminiscent of what was elsewhere observed with the system double stranded RNA-seminal RNAase (also a very basic, dimeric enzyme), and could, therefore, tentatively be correlated with the dimeric and/or the very basic nature of the enzyme protein.     

No salting-in of lysozyme chloride observed at low ionic strength over a large range of pH.

Solubility of lysozyme chloride was determined in the absence of added salt and in the presence of 0.05-1.2 M NaCl, starting from isoionic lysozyme, which was then brought to pH values from 9 to 3 by addition of HCl. The main observation is the absence of a salting-in region whatever the pH studied. This is explained by a predominant electrostatic screening of the positively charged protein and/or by adsorption of chloride ions by the protein. The solubility increases with the protein net charge at low ionic strength, but the reverse is observed at high ionic strength. The solubility of lysozyme chloride seems to become independent of ionic strength at pH approximately 9.5, which is interpreted as a shift of the isoionic pH (10.8) to an isoelectric pH due to chloride binding. The crystallization at very low ionic strength, where lysozyme crystallizes at supersaturation values as low as 1.1, amplifies the effect of pH on protein solubility. Understanding the effect of the net charge and of ionic strength on protein-protein interactions is valuable not only for protein crystal growth but more generally also for the formation of protein-protein or protein-ligand complexes.     

The effect of pH and ionic strength on the electrophoretic separation of acidic glycosaminoglycans

Using the electrophoretical methods applied to this study it is possible to determinate the dissociation constants (pK) of acid glycosaminoglycans containing a carboxylic group. The pK-values of the six acid glycosaminoglycans separated from animal connective tissues determined in this work were: hyaluronic acid (HA), pK = 3.0; chondroitin sulfate A (CS-A), pK = 2.8; chondroitin sulfate C (CS-C), pK = 3.3; dermatan sulfate (CS-B), pK = 3.3; heparatin sulfate (HeS), pK = 3.1 and heparin (HeP), pK = 2.4 and were measured at a constant ionic strength of I = 0.164 (NaCl) and at 10 ± 2°C.Variation of ionic strength showed that physiological conditions seem to be most suitable for the electrophoretic separation of the glycosaminoglycans studied. A decrease of ionic strength causes increasing mobility but less accurate spots. In the case of increasing ionic strength the results are vice versa.The second spot for HA very often appeared when pH values higher than 2 were used for electrophoresis. The spot had the same form as the original, high intensity, but an undecided migration in the pH range near the pK value of HA (3.0).     

D2O-induced ion channel activation in Characeae at low ionic strength

Effects of D₂O were studied on internodal cells of the freshwater alga Nitellopsis obtusa under plasmalemma perfusion (tonoplast-free cells) with voltage clamp, and on Ca²⁺ channels isolated from the alga and reconstituted in bilayer lipid membranes (BLM). External application of artificial pond water (APW) with D₂O as the solvent to the perfused plasmalemma preparation led to an abrupt drop of membrane resistance (R _m = 0.12 ±0.03 kΩ · cm²), thus preventing further voltage clamping. APW with 25% D₂O caused a two-step reduction of R _m : first, down to 2.0 ± 0.8 kΩ · cm², and then further to 200 Ω · cm², in 2 min. It was shown that in the first stage, Ca²⁺ channels are activated, and then, Ca²⁺ ions entering through them activate the Cl^? channels. The Ca²⁺ channels are activated irreversibly. If 100 mm CsCl was substituted for 200 mm sucrose (introduced for isoosmoticity), no effect of D₂O on R _m was observed. Intracellular H₂O/D₂O substitution also did not change R _m . In experiments on single Ca²⁺ channels in BLM H₂O/ D₂O substitution in a solution containing 100 mm KCl (trans side) produced no effect on channel activity, while in 10 mm KCl, at negative voltage, the open channel probability sharply increased. This effect was irreversible. The single channel conductance was not altered after the H₂O/D₂O substitution. The discussion of the possible mechanism of D₂O action on Ca²⁺ and Cl^? channels was based on an osmotic-like stress effect and the phenomenon of higher D-bond energy compared to the H-bond.     

Effects of alkaline pH on sarcoplasmic reticulum Ca2+ release and Ca2+ uptake.

Alkalinization-induced Ca2+ release from isolated frog or rabbit sarcoplasmic reticulum vesicles appears to consist of two distinct components: 1) a direct activation of ruthenium red-sensitive Ca2+ release channels in terminal cisternae and 2) an increased ruthenium red-insensitive Ca2+ efflux through some other efflux pathway distributed throughout the sarcoplasmic reticulum. The first of these releases exhibits an alkalinization-induced inactivation process and does not depend on the ruthenium red-insensitive form of Ca2+ release as a triggering agent for secondary Ca(2+)-induced Ca2+ release. Both releases are inhibited when the extravesicular (i.e. cytoplasmic) free [Ca2+] is reduced. This may reflect an increased sensitivity of the Ca2+ release channels to Ca2+ at alkaline pH. The pH sensitivity of the ruthenium red-sensitive Ca2+ release channels could be of significance during excitation-contraction coupling. The ruthenium red-insensitive form of Ca2+ release is less likely to be physiologically relevant, but it probably has contributed greatly to reports of alkalinization-induced decreases in net sarcoplasmic reticulum Ca2+ uptake, particularly under conditions where oxalate supported Ca2+ uptake is much less affected, as here.     

Rabbit skeletal muscle F-actin can be stable at low ionic strength, provided trace amounts of Ca2+ are absent.

Addition of low concentrations (0.2--2.0 mM) of EGTA to rabbit skeletal muscle G-actin in the presence of ATP caused increase in viscosity. The effect is probably due to chelation of Ca2+. EGTA-polymerized actin was sedimented in the ultracentrifuge as a pellet which could be depolymerized in the presence of Ca2+ and then repolymerized. Electron microscopy indicated that formation of filamentous actin which appears to be somewhat more flexible than F-actin obtained by polymerization with KCl. The EGTA-polymerized actin was dissociated by DNAase I faster than KCl-polymerized actin. F-Actin can thus be stable also in very low ionic strength media if Ca2+ is removed whereas for G-actin to be the only form of the protein in such media, micromolar concentrations of Ca2+ must be present.     

Reduced Ca(2+)-induced Ca2+ release from skeletal muscle sarcoplasmic reticulum at low pH.

The purpose of this investigation was to determine the effects of reduced pH on Ca(2+)-induced Ca2+ release (CICR) from skeletal muscle sarcoplasmic reticulum (SR). Frog semitendinosus fiber bundles (1-3/bundle) were chemically skinned via saponin treatment (50 micrograms/mL, 20 min), which removes the sarcolemma and leaves the SR functional. The SR was first depleted of Ca2+ then loaded for 2 min at pCa (log free Ca2+ concentration) 6.6. CICR was then evoked by exposing the fibers to pCa 5-7 for 5-60 s. CICR was evoked both in the absence of ATP and Mg2+ and in the presence of beta, gamma-methyleneadenosine-5'-triphosphate (AMPPCP, a nonhydrolyzable form of ATP) and Mg2+. Ca2+ remaining in the SR was then assayed via caffeine (25 mM) contracture. In all cases, CICR evoked at pH 6.5 resulted in larger caffeine contractures than that evoked at 7.0, suggesting that more Ca2+ was released during CICR at the higher pH. Accordingly, rate constants for CICR were significantly greater at pH 7.0 than at pH 6.5. These results indicate that reduced pH depresses CICR from skeletal muscle SR.