Deamidation of glutaminyl residues: dependence on pH, temperature, and ionic strength

We have shown the dependence of the deamidation half-times of the peptides, GlyLeuGlnAlaGly and GlyArgGlnAlaGly upon pH, temperature, and ionic strength. Increase in temperature or ionic strength, variation of pH to pH′s higher or lower than pH 6, and the use of phosphate buffer rather than Tris buffer at high pH all decrease the half-time of dcamidation. Temperature increase of 20°C or pH change of 2 pH units decreases the half-time about fivefold, while increase of one ionic strength unit decreases the half-time about twofold. In pH 7.4, I = 0.2, 37.0°C phosphate buffer, the deamidation half-times are 663 ± 74 and 389 ± 56 days respectively for the two peptides, GlyLeuGlnAlaGly and GlyArgGlnAlaGly.These experiments should serve as a warning to peptide and protein experimenters that even the more stable glutaminyl residues are unstable with respect to deamidation in certain solvent conditions. These experiments also provide, along with previously reported experiments on asparaginyl peptides (7), some quantitative data to help with the extrapolation of in vitro deamidation experiments to in vivo deamidation conditions.     

Protein unfolding in detergents: effect of micelle structure,ionic strength,pH, and temperature

The 101-residue monomeric protein S6 unfolds in the anionic detergent sodium dodecyl sulfate (SDS) above the critical micelle concentration, with unfolding rates varying according to two different modes. Our group has proposed that spherical micelles lead to saturation kinetics in unfolding (mode 1), while cylindrical micelles prevalent at higher SDS concentrations induce a power-law dependent increase in the unfolding rate (mode 2). Here I investigate in more detail how micellar properties affect protein unfolding. High NaCl concentrations, which induce cylindrical micelles, favor mode 2. This is consistent with our model, though other effects such as electrostatic screening cannot be discounted. Furthermore, unfolding does not occur in mode 2 in the cationic detergent LTAB, which is unable to form cylindrical micelles. A strong retardation of unfolding occurs at higher LTAB concentrations, possibly due to the formation of dead-end protein-detergent complexes. A similar, albeit much weaker, effect is seen in SDS in the absence of salt. Chymotrypsin inhibitor 2 exhibits the same modes of unfolding in SDS as S6, indicating that this type of protein unfolding is not specific for S6. The unfolding process in mode 1 has an activation barrier similar in magnitude to that in water, while the activation barrier in mode 2 is strongly concentration-dependent. The strong pH-dependence of unfolding in SDS and LTAB suggests that the rate of unfolding in anionic detergent is modulated by repulsion between detergent headgroups and anionic side chains, while cationic side chains modulate unfolding rates in cationic detergents.     

Hydrophobicity and helicity of membrane-interactive peptides containing peptoid residues

Peptoid (N-alkylglycyl) residues in peptides have been studied in a variety of applications, but their behavior in membrane environments has not been systematically investigated. We have synthesized a series of membrane-interactive peptides of prototypic structure KKAAAXAAAAAXAAWAAXAAAKKKK-amide, where X corresponds to the peptoid residues Nala (= sarcosine), Nval, Nile, Nleu, Nphe, and Ntrp. Investigation of their relative hydrophobic character by high-performance liquid chromatography indicated an order of hydrophobicity Ntrp > Nphe > Nleu > Nile > Nval > Nala-largely parallel to the relative scale for these side-chains in natural amino acids, although all values were significantly more "hydrophilic" than their amino acid correspondents. Conformations of peptoid-containing peptides measured by circular dichroism spectroscopy were unordered in the presence of SDS micelles but helical for peptides with X = the corresponding amino acids, suggesting a general helix-breaking tendency for the peptoid residues. However, peptides were able to form helical structures in the solvent n-butanol, indicating that this conformation is possible if peptides became inserted into micellar phases. The latter notion was confirmed by increasing hydrophobic content of the peptides by embedding peptoid Nala residues in Leu-rich rather than Ala-rich sequences, which promoted peptide insertion and helical structure in micelles. The overall results suggest that judicious interspersing of amino acid and peptoid residues in peptide sequences can produce hydrophobic water-soluble materials with membrane-partitioning capacity.     

The transient complex of poplar plastocyanin with cytochrome f: effects of ionic strength and pH

The orientation of poplar plastocyanin in the complex with turnip cytochrome f has been determined by rigid-body calculations using restraints from paramagnetic NMR measurements. The results show that poplar plastocyanin interacts with cytochrome f with the hydrophobic patch of plastocyanin close to the heme region on cytochrome f and via electrostatic interactions between the charged patches on both proteins. Plastocyanin is tilted relative to the orientation reported for spinach plastocyanin, resulting in a longer distance between iron and copper (13.9 A). With increasing ionic strength, from 0.01 to 0.11 M, all observed chemical-shift changes decrease uniformly, supporting the idea that electrostatic forces contribute to complex formation. There is no indication for a rearrangement of the transient complex in this ionic strength range, contrary to what had been proposed earlier on the basis of kinetic data. By decreasing the pH from pH 7.7 to pH 5.5, the complex is destabilized. This may be attributed to the protonation of the conserved acidic patches or the copper ligand His87 in poplar plastocyanin, which are shown to have similar pK(a) values. The results are interpreted in a two-step model for complex formation.     

Steady-state properties of calcium binding to parvalbumins from bullfrog skeletal muscle: effects of Mg2+, pH, ionic strength, and temperature

To improve our understanding of the physiological roles of parvalbumins, PA-1 (pI 4.78) and PA-2 (pI 4.97) parvalbumins were prepared from bullfrog skeletal muscle and their calcium binding properties were examined in a medium of constant ionic strength (I = 0.106, pH 6.80, at 20 degrees C) containing various concentrations of Mg2+ by using a metallo-indicator, tetramethylmurexide. Apparent binding constants for Ca2+ in the presence of Mg2+ changed in the manner expected if Ca2+ and Mg2+ compete for two independent homogeneous binding sites. The following values were obtained: for PA-1, KCa = 1 X 10(7) M-1, KMg = 900 M-1; for PA-2, KCa = 6 X 10(6) M-1, KMg = 830 M-1 (I = 0.106, pH 6.80, at 20 degrees C). The apparent binding constants are strongly dependent on temperature: at 10 degrees C for PA-1, KCa = 2 X 10(8) M-1, KMg = 10(4) M-1; for PA-2, KCa = 5 X 10(7) M-1, KMg = 5 X 10(3) M-1 (I = 0.106, pH 6.80). The dependence of the affinities for Ca2+ on ionic strength is similar to or less than that of GEDTA (EGTA). The affinities for Ca2+ and Mg2+ of parvalbumins are unchanged between pH 6.5 and 7.2.     

Hydrophobic surface properties of myosin in solution as studied by partition in aqueous two-phase systems: effects of ionic strength,pH and temperature

Summary The effect of gonadectomy (at the 10th day of life) and treatment with sexual steroids (during the first month) upon development of alpha-amylase activity in rat parotid gland has been studied.Alpha-amylase specific activity of parotid glands from 20-day-old orchidectomized rats and from 25-day-old ovariectomized animals was significantly higher than that of intact male and female rats of the same age respectively. Spayed males treated with testosterone (10 g/day on the 13th, 15th, and 17th day) and ovariectomized rats treated with oestradiol (2.5 g/day from the 16th to the 22nd day) showed values of enzymic activity similar to those of normal animals.Results indicate that oestradiol and testosterone have an inhibitory effect upon the increase of alpha-amylase activity in parotid gland during a very defined period of development.Career investigators of Consejo Nacional de Investigaciones Cientificas y Técnicas.     

Swelling studies on the cornea and sclera: the effects of pH and ionic strength.

The biophysical properties of the cornea and sclera depend on the precise maintenance of tissue hydration. We have studied the swelling of the tissues as a function of pH and ionic strength of the bathing medium, using an equilibration technique that prevents the loss of proteoglycans during swelling. Synchrotron x-ray diffraction was used to measure the average intermolecular and interfibrillar spacings, the fibril diameters, and the collagen D-periodicity. We found that both tissues swelled least near pH 4, that higher hydrations were achieved at lower ionic strengths, and that sclera swelled about one-third as much as cornea under most conditions. In the corneal stroma, the interfibrillar spacing increased most with hydration at pH values near 7. Fibril diameters and D-periodicity were independent of tissue hydration and pH at hydrations above 1. Intermolecular spacings in both tissues decreased as the ionic strength was increased, and there was a significant difference between cornea and sclera. Finally, we observed that corneas swollen near pH 7 transmitted significantly more light than those swollen at lower pH levels. The results indicate that the isoelectric points of both tissues are close to pH 4. The effects of ionic strength can be explained in terms of chloride binding within the tissues. The higher light transmission achieved in corneas swollen at neutral pH may be related to the fact that the interfibrillar fluid is more evenly distributed under these conditions.     

Transport of natural soil nanoparticles in saturated porous media: effects of pH and ionic strength

To understand the effects of ionic strength and pH on the transport of natural soil nanoparticles (NS) in saturated porous media, aeolian sandy soil nanoparticles (AS), cultivated loessial soil nano particles (CS), manural loessial soil nanoparticles (MS) and red soil nanoparticles (RS) were leached with solutions of varying pH and ionic strength. The recovery rate of soil nanoparticles decreased in the order AS > RS > MS > CS. Transport of soil nanoparticles was enhanced with increasing pH and decreasing ionic strength and was attributable to changes in the Zeta potential of NS. Deposition of NS was also affected by the composition of soil nanoparticles and the surface charge. Column experiments showed that the interaction between soil nanoparticles and saturated quartz sand was mainly due to the physical and chemical properties of soil nanoparticles. The Derjaguin–Landau–Verwey–Overbeek interaction energies between NS and sand were affected by pHs and ionic strengths. Soil nanoparticles transport through saturated porous media could be accurately simulated by the one-dimensional advection-dispersion-reaction equation.     

Buffer, pH, and ionic strength effects on the (Na+ + K+)-ATPase

A dog kidney (Na+ + K+)-ATPase preparation also catalyzes K+-independent and K+-activated phosphatase reactions with p-nitrophenyl phosphate as substrate. K+-independent activity increases with declining pH over the range 7.5 to 5.8, whereas the other two activities decrease. The increased K+-independent activity is similar with imidazole, histidine, and several Good buffers, and is thus attributable to free H+, probably by affecting enzyme conformations rather than by changing affinity for Mg2+ or substrate or by H+ occupying specific K+-sites. The decrease in K+-phosphatase and (Na+ + K+)-ATPase activities with pH also occurs similarly with those buffers, and is not due to changes in apparent affinity for substrate or for cation activators. However, the Good buffers Pipes and ADA inhibit the K+-independent phosphatase reaction strongly, the K+-activated reaction moderately, and the (Na+ + K+)-ATPase reaction little; both contain two acidic groups, unlike the other buffers tested. Inhibition of the phosphatase reaction by Pipes is associated with a decreased apparent affinity for K+ and an increased sensitivity to inhibition by Na+ and ADP, consistent with Pipes hindering conformational transitions to the E2 enzyme forms required for phosphatase hydrolytic activity.     

Cartilage electromechanics--I. Electrokinetic transduction and the effects of electrolyte pH and ionic strength

Articular cartilage contains a high fixed charge density under physiological conditions associated primarily with the ionized proteoglycan molecules of the extracellular matrix. Oscillatory compression of cartilage using physiological loads produces electrical potentials that have been shown previously to be the result of an electrokinetic (streaming) transduction mechanism. We have now observed two additional electromechanical phenomena not previously seen in cartilage or other soft tissues: 'streaming current' and 'current-generated stress'. Sinusoidal mechanical compression induced a sinusoidal streaming current density through cartilage disks when the Ag/AgCl electrodes that were used to compress the cartilage were shorted together externally. Conversely, a sinusoidal current density applied to the tissue generated a sinusoidal mechanical stress within the tissue. Both these phenomena were found to be consistent with the same electrokinetic transduction mechanism responsible for the streaming potential. Changes in the measured streaming potential response that resulted from modification of bath ionic strength and pH have provided additional insights into the molecular origins of these transduction processes. Finally, we have now observed streaming potentials in living cartilage maintained in organ culture, as well as in previously frozen tissue.     

The effects of pH and ionic strength on the partitioning of four proteins in reverse micelle systems

Four proteins with different physicochemical properties have been partitioned in reversed micelle systems: thaumatin, ribonuclease A, soybean trypsin inhibitor, and alpha-lactalbumin. The organic phase was formed by sodium salt (AOT) in isooctane, and the aqueous phase contained KCl, KBr, MgCl(2), or NaCl. Aqueous phase pH was varied between 2 and 13 and ionic strength from 0.1 to 1.0 M. Small changes in pH [around the isoelecric point (pl)] were found to influence the solubilization of ribonuclease A and trypsin inhibitor, but for thaumatin the pH change necessary to affect partition was much greater as a consequence of the difference in net charge (titration curves) of these protein molecules as pH changes. The type of ions present in the system was also a determining factor for partition; the larger ions (K(+)) produced more electrostatic screening and hence less protein solubilization than the smaller ions (Na(+)). With changes in ionic strength surface hydrophobicity was a dominant factor affecting solubilization of thaumatin in NaCl-containing systems at high pH. Charge distribution and hydrophobicity are thought to be important parameters when partitioning the protein alpha-lactalbumin. (c) 1994 John Wiley & Sons, Inc.     

Calcium binding to troponin C and troponin: effects of Mg2+, ionic strength and pH

Calcium binding to troponin C and troponin was examined by a metallochromic indicator method under various conditions to obtain a further understanding of the regulatory roles of these proteins in muscle contraction. Troponin C has four Ca binding sites, of which 2 sites have a high affinity of 4.5 X 10(6) M-1 for Ca2+ and the other 2 sites have a low affinity of 6.4 X 10(4) M-1 in a reaction medium consisting of 100 mM KCl, 20 mM MOPS-KOH pH 6.80 and 0.13 mM tetramethylmurexide at 20 degrees C. Magnesium also binds competitively to both the high and low affinity sites: the apparent binding constants are 1,000 M-1 and 520 M-1, respectively. Contrary to the claim by Potter and Gergely (J. Biol. Chem. 250, 4628-4633, 1975), the low affinity sites are not specific only for Ca2+. The high and low affinity sites of troponin C showed different dependence on the ionic strength: the high affinity sites were similar to GEDTA, while the low affinity sites were similar to calmodulin, which has a steeper ionic strength dependence than GEDTA. Ca binding to troponin C was not affected by change of pH between 6.5 and 7.2. Troponin I enhanced the apparent affinity of troponin C for Ca2+ to a value similar to that for troponin. Trifluoperazine also increased Ca binding to troponin C. Troponin has four Ca binding sites as does troponin C, but the affinities are so high that the precise analysis was difficult by this method. The apparent binding constants for Ca2+ and Mg2+ were determined to be 3.5 X 10(6) M-1 and 440 M-1, respectively, for low affinity sites under the same conditions as for troponin C, being independent of change in pH between 6.5 and 7.2. The competitive binding of Mg2+ to the low affinity sites of troponin is consistent with the results of Kohama (J. Biochem. 88, 591-599, 1980). The estimate for the high affinity sites is compatible with the reported results.     

Effect of temperature,pH and ionic strength on hydroxyapatite stabilised Pickering emulsions produced in batch and continuous mode

Oil-in-water (O/W) Pickering emulsions are attracting attention as carriers of lipophilic active compounds with clear advantages over traditional systems. Having in view their effective use it is important to study their stability against environmental stresses impacting manufacture, storage, and application conditions. In this work, hydroxyapatite nanoparticles (n-HAp) Pickering emulsions produced in continuous mode using a mesostructured reactor (average size?~?7, 11 and 18 µm) and in batch mode using a rotor–stator device (average size?~?18 µm) were studied concerning their behaviour at different temperatures (5–90 ºC), pH (2–10) and ionic strength (0–500 mM), conditions with relevance for food applications. Droplet size, morphology, and zeta-potential were analysed after 1 and 7 days under storage. In general, and despite the droplet size, the n-HAp Pickering emulsions were stable within the tested ionic strength range, at relatively high pH environments (6–10), and at temperatures up to 70 ºC. Pickering emulsions undergo complete phase separation at very low pH (2) due to n-HAp particle's disruption. A clear tendency to aggregation and coalescence was observed for high temperatures (70–90 ºC). Results indicate no significant differences related to the used production method. From an industrial perspective, this work also corroborates that the scale-up to a continuous process using a mesostructured reactor, NETmix, from a batch laboratorial process is feasible without impacting stability.

Graphical abstract

     

1,2-Dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG) monolayers: influence of temperature, pH, ionic strength and binding of alkaline earth cations

Ion binding and lipid ionization of the acidic phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG) in monolayers was studied by measuring the lateral pressure Pi as a function of the molecular area A at the air/water interface at different temperatures. The pH of the subphase (pH 2 and 7) and the ionic strength (NaCl) was varied. In addition, different divalent cations (1mM MgCl2, CaCl2 and SrCl2, pH 7) were added. DMPG is partly protonated on pure water at pH 7. An increase in the NaCl concentration in the subphase leads to film expansion. This effect is caused by an ionization of the headgroup of DMPG, i.e. a shift of the apparent pK. More condensed films are obtained on pure water at pH 2, due to the reduction of electrostatic repulsion by headgroup protonation and the possibility for the formation of a hydrogen bonding network. The divalent cations Mg2+, Ca2+ and Sr2+ interact differently with a DMPG monolayer in pure water at pH 7. In the presence of 1mM CaCl2 a condensation of the DMPG film is induced, whereas an expansion of the monolayer is observed in the presence of Mg2+ and Sr2+. Two counteracting effects are operative: (a) ionization of the headgroup due to electrostatic screening leads to film expansion and (b) binding of the divalent cations to the lipid headgroups leads to condensation. The latter effect is more pronounced in the case of Ca2+, whereas the binding of Mg2+ and Sr2+ to DMPG is weaker. Site-specific cation binding has to be assumed in addition to electrostatic effects.