Hydrogen-deuterium exchange of a primary amide as a model for asparagine and glutamine exchange in proteins

Hydrogen-deuterium exchange of the primary amide, isobutyramide, was investigated as a model for asparagine and glutamine-NH₂ exchange in a protein. A simple amide was chosen since the structures of several well-characterized proteins show most of these residues to be exposed to solvent. Isobutyramide-exchange data were obtained in 1:1 D₂O:dioxane solutions using a near-infrared method. The rate data were strictly pseudo-first order and yielded an average of 95% exchange of the primary amide hydrogens. In analogy with secondary amides, the pD dependence of the rate constants was characteristic of specific acid and base catalysis. In addition, analysis of the rate-pD profile for isobutyramide indicated a significant uncatalyzed exchange reaction. Temperature-dependence studies of the first-order rate constants at a fixed pD yielded an apparent activation energy of 19.3 kcal/mole. Predicted half-life times for the exposed primary amide hydrogens in proteins, based on these exchange parameters, indicate that asparagine and glutamine side chains generally would contribute to the overall rate data only below 15°C and then only for approximately 1 pD unit around the point of minimum reaction velocity.     

A two-dimensional NMR study of exchange behavior of amide hydrogens in a lysozyme derivative with an extra cross-link between Glu35 and Trp 108—quenching of cooperative fluctuations and effects on the protein stability

Two dimensional nmr spectra [correlated spectroscopy (COSY), homonuclear Hartmann-Hahn (HOHAHA), nuclear Overhauser effect spectroscopy (NOESY)] have been observed for cross-linked lysozyme, a chemically modified lysozyme derivative with an extra ester cross-link between residues E35 and W108. Eight shifted cross-peaks were found in the fingerprint region of COSY spectra. By searching COSY, HOHAHA and NOESY spectra, they have been assigned to A32, E35, S36, I58, A107, W108, V109, and A110. The NOE connectivities (d_NN and d_αN) found for the cross-linked lysozyme are quite similar to those for the intact lysozyme. Exchange behavior of amide hydrogens has been studied for both intact and cross-linked lysozymes by observing the fingerprint region of COSY spectra. Hydrogen exchange reactions were carried out at pH 7.0 and at several temperatures. There exist 41 amide hydrogens whose exchange reactions are detectable under this experimetal conditon. Not only exchange rates but also their activation enthalpies were determined for individual amide hydrogens. They are classified into two groups, which are called categories III and IV. Category III hydrogens are distributed in relatively flexible peripheral parts of protein, and category IV hydrogens are deeply buried in the core region of protein. Category III hydrogens are exchanged through localized unfolding around their sites with a low activation enthalpy ranging from 10 to 25 kcal/mol. The formation of an extra cross-link affects neither the exchange rate nor the activation enthalpy of category III hydrogens. However, amide hydrogens of residues 34–39 in the vicinity of the hinge are exceptions. They are easily exchanged in the intact lysozyme but their exchange rates are drastically retarded by cross-linking. In the intact lysozyme, structural fluctuations mediating the exchange of category IV hydrogens are highly cooperative with a large activation enthalpy. These large-scale structural fluctuations are the global unfolding of the overall structure and also concerted motions within a domain. Especially near 38°C, it was found that the dominant fluctuation occurring in the α-domain is different from that in the β-domain. However, these concerted motions are strongly quenched by the formation of the cross-link because of the cooperativity of such a large-scale fluctuation. The stabilization of a localized area of protein by cross-linking results in the great suppression of large-scale and concerted motions. The exchange rates of category IV hydrogens are extremely retarded in the cross-linked lysozyme, so that they are exchanged through the so-called penetration mechanism characterized by a low activation enthalpy. These expeimental results are discussed with regard to the contribution of cross-linking to the stabilization of the folded structure of protein. © 1997 John Wiley & Sons, Inc.     

Glycine transport by hemolysed and restored pigeon red cells effects of a domnan-induced electrical potential on entry and exit kinetics

The influence of a Donnan effect on the transport of glycine by hemolysed and restored pigeon red cells was examined. The Donnan effect was produced by replacing Cl^? with 2,4-toluenedisulfonate or glutamate. The effects of the associated membrane potential and inside-outside pH difference on glycine entry and exit rates were examined. The effects of pH on entry and exit rates in the absence of a Donnan effect were also examined.In the absence of a Donnan effect, Na⁺-dependent glycine entry requires the protonated form of a group with a pK_app of 7.9 and the depronated form of another group with a pK_app of 6.8. Neither of these are required for exit but the deprotonated form of a group(s) with a pK_app of 6.2 is required. The pK 7.9 group and pK 6.2 group probably react with H⁺ at the inner face of the membrane and the pK 6.8 group probably reacts at the outer face.The V for glycine entry was determined for cells with their Cl^? largely replaced by toluenedisulfonate and without such replacement. Between pH 6.1 and 7, the ratio of the respective V values, V_T/V_Cl, was 1.5–1.7. V_T/V_Cl rose above pH 7 to near 4 at pH 8.3. At pH 6.9, with glutamate replacing cell Cl^?, the analogous ratio (V_Glu/V_Cl) was 1.7. The increase of V_T/V_Cl above pH 7 could be quantitatively accounted for by the increase in cell [H⁺]/medium [H⁺] caused by the Donnan effect together with the assumption that the pK 7.9 group reacts with H⁺ at the inner face of the membrane.When cell Cl^? was replaced by toluenedisulfonate or glutamate there was a drop in the term in the glycine K_m describing Na⁺ dependence of glycine entry. When cell Cl^? was replaced by toluenedisulfonate there was a rise in the Na⁺-independent term in the glycine entry K_m. By replacing varying amounts of cell Cl^? with either toluenedisulfonate or glutamate, plots were obtained of entry rates vs. the cell [Cl^?]/medium [Cl^?] ratio consistent with the assumption that the Donnan-induced membrane potential acts on a “moving” charge. Glycine exit was only slightly accelerated by trans-toluenedisulfonate. The ratio, exit rate into toluenedisulfonate medium/exit rate into Cl^? medium rose with decreasing pH. This rise could be accounted for by a Donnan-induced inside-outside pH difference which affects a pK_app 6.2 group reacting with internal H⁺.The observed influences of the Donnan effect on V(glycine entry), on both components of K_m(glycine entry), on the shape of the plot of glycine entry rate vs. the cell [Cl^?]/medium [Cl^?] ratio and on glycine exit all fit the assumptions that when the empty porter reorients, one unit of negative charge accompanies it “across” the membrane and that no other steps involve charge movement.The properties of the system seem inconsistent with a translational (“ferry boar”) mobile carrier.     

Étude de l'interaction de la thiamine diphosphate avec l'ion magnésium

The action of magnesium ion on the exchange rate of the proton in C₂ of thiamine and thiamine diphosphate is studied at different values of pD. Above pD 5 the ion Mg²⁺ increases this exchange rate. The phenomenon is markedly enhanced for TDP rather than thiamine and increases with pD. Below pD 5 magnesium decreases the exchange rate. This decrease is greater for TDP than for thiamine. The maximum effect is reached at a magnesium concentration of 0.5/1 for thiamine and of 1/1 for TDP.T₁ measurements are made for different pH values with and without magnesium ion. Results seem to prove that an increase in pD values from 3.9 to 5.9 leads to an accentuation of the molecules «folded form. Nevertheless for a given pD value the TDP-Mg complex seems to have a more «folded form than TDP.     

H Cotransports in Corn Roots as Related to the Surface pH Shift Induced by Active H Excretion

The surface pH shift induced by active H⁺ excretion in corn (Zea mays L.) roots was estimated using acetic acid influx as a pH probe (H Sentenac, C Grignon 1987 Plant Physiol 84: 1367-1372). At constant bulk pH, buffering the medium strongly reduced the magnitude of the surface pH shift. This was used to study the effect of surface pH shift on H⁺ cotransports. In the absence of buffers, the surface pH shift increased with the bulk pH. Buffers decreased ³²Pi influx and this effect was stronger at pH 7.2 than at pH 5.8, and stronger in the absence than in the presence of an inhibitor of the proton pump (vanadate). Buffers exerted a similar depressive and pH-dependent effect on net NO₃⁻ uptake. They hyperpolarized the cell membrane, and stimulated ⁸⁶Rb⁺ influx, K⁺:H⁺ net exchange, and malate accumulation. These results are consistent with the hypothesis that H⁺ accumulation at the cell surface is effective in driving H⁺ reentry. We concluded that the surface pH shift due to proton pump activity is involved in the energetic coupling of H⁺ cotransports.     

Solubility of lysozyme in polyethylene glycol-electrolyte mixtures: the depletion interaction and ion-specific effects

The solubility of aqueous solutions of lysozyme in the presence of polyethylene glycol and various alkaline salts was studied experimentally. The protein-electrolyte mixture was titrated with polyethylene glycol, and when precipitation of the protein occurred, a strong increase of the absorbance at 340 nm was observed. The solubility data were obtained as a function of experimental variables such as protein and electrolyte concentrations, electrolyte type, degree of polymerization of polyethylene glycol, and pH of the solution; the last defines the net charge of the lysozyme. The results indicate that the solubility of lysozyme decreases with the addition of polyethylene glycol; the solubility is lower for a polyethylene glycol with a higher degree of polymerization. Further, the logarithm of the protein solubility is a linear function of the polyethylene glycol concentration. The process is reversible and the protein remains in its native form. An increase of the electrolyte (NaCl) concentration decreases the solubility of lysozyme in the presence and absence of polyethylene glycol. The effect can be explained by the screening of the charged amino residues of the protein. The solubility experiments were performed at two different pH values (pH = 4.0 and 6.0), where the lysozyme net charge was +11 and +8, respectively. Ion-specific effects were systematically investigated. Anions such as Br⁻, Cl⁻, F⁻, and (all in combination with Na⁺), when acting as counterions to a protein with positive net charge, exhibit a strong effect on the lysozyme solubility. The differences in protein solubility for chloride solutions with different cations Cs⁺, K⁺, and Na⁺ (coions) were much smaller. The results at pH = 4.0 show that anions decrease the lysozyme solubility in the order (the inverse Hofmeister series), whereas cations follow the direct Hofmeister series (Cs⁺ < K⁺ < Na⁺) in this situation.     

Changes in Chloride Fluxes and Cytosolic pH Induced by Abscisic Acid in Elodea densa Leaves

The effects of ABA on intracellular pH, net H⁺ extrusion, Cl^? fluxes and E_m values were studied in Elodea densa leaves, and the possible relationships between the ABA-induced changes of cytosolic pH and of Cl^? and H⁺ fluxes were investigated. Cytosolic and vacuolar pH were calculated by the weak acid and weak base distribution method. The data show that, also in this material (a water plant without stomata), ABA induces a decrease in both net H⁺ extrusion and intracellular pH, and strongly inhibits Cl^? efflux. No significant effect of ABA is detectable on E_m values, either at short or long intervals in the presence or absence of K⁺. Cl^? efflux is apparently independent of the activity of the plasmalemma H⁺ pump and of the E_m values. Conversely, it strongly depends on the value of cytosolic pH, a larger efflux occurring for the lower pH values both in the presence and in the absence of ABA. These results indicate that the ABA-induced cytosolic acidification cannot be the cause but, possibly, a consequence of the decrease in Cl^? efflux, and are consistent with the hypothesis of a primary role of ABA in regulating Cl^? efflux, presumably by directly affecting a class of Cl^?-permeable channels.     

The role of protonation in protein fibrillation

Many proteins fibrillate at low pH despite a high population of charged side chains. Therefore exchange of protons between the fibrillating peptide and its surroundings may play an important role in fibrillation. Here, we use isothermal titration calorimetry to measure exchange of protons between buffer and the peptide hormone glucagon during fibrillation. Glucagon absorbs or releases protons to an extent which allows it to attain a net charge of zero in the fibrillar state, both at acidic and basic pH. Similar results are obtained for lysozyme. This suggests that side chain pK_a values change dramatically in the fibrillar state.     

cAMP activates Cl−/HCO3− exchange for regulation of intracellular pH in renal epithelial cells

The role of cAMP in regulation of intracellular pH in the confluent LLC-PK₁ cells was investigated. DibutyrylcAMP and forskolin induce intracellular acidification. This acidification is inhibited by DIDS and ethacrynic acid, inhibitors of Na⁺-independent Cl^?/HCO₃^? exchange, and by removal of extracellular Cl^?. In addition, Bt₂ cAMP causes Cl^? entry into LLC-PK₁ cells. These results suggest that cAMP activates Cl^? transport, namely Na⁺-independent Cl^?/HCO₃^? exchange, which participates in pH_i regulation.     

Determining the net flux of charge released by maize roots by directly measuring variations of the alkalinity in the nutrient solution

The net flux of charge released by maize, i.e. the strong ion exchange balance between the roots and their environment, was determined in acidic and alkaline solutions, i.e. solutions with a low and a high pH buffering capacity, respectively. The work was based on direct measurement of total alkalinity in culture solutions over a period of several days.The results show there was little difference in the net flux of charge released by maize in acidic and alkaline solutions: In both cases, approximately –1 mol_c (kg DM)^–1 s^–1. As the maize was grown in a non-limiting nitrate solution, the charge flux was negative, corresponding to a net release of hydroxyls into the rhizosphere. In contrast, the change in the amounts of free protons in the solution was approximately 1 nmol (kg DM)^–1 s^–1, i.e. 3 orders of magnitude lower than the net charge flux. Moreover, it was negative in acidic media , i.e. the solution pH increased, and positive in alkaline media, i.e. the solution pH decreased. This decrease probably resulted from the release of inorganic carbon by the roots. The effect on the change in solution pH was only slight in acidic conditions but considerable in alkaline conditions, where it reduced the pH even though the culture solution was alkalinised by the roots.The difference in the way that acidic and alkaline solutions function demonstrates the importance of the pH buffering capacity of the solution in determining the net flux of charge released by the plants. It underlines the difficulty of estimating the net charge flux from pH change measurements in the rhizosphere.     

Effects of pH changes and charge characteristics in the uptake of norepinephrine by synaptosomes of rat brain

The pH dependence of the initial uptake of norepinephrine by rat whole brain synaptosomes was studied short incubation times at 37°C in order to examine the possible involvement of the phenolic OH group. The pH vs. uptake profile exhibits a maximum near pH 8.2 in H₂O medium. When the medium was changed to ²H₂O, the profile showed a shift of maximum corresponding to the pK_a change of the phenolic OH group. The pH vs. uptake profile of tyramine was quite different from that of norepinephrine. These pH effects on uptake were explained as manifestations of the involvement of the phenolic OH group in the process.The amine and phenolic hydroxyl groups in norepinephrine were studied separately by employing two series of compounds structurally related to catecholamines, amphetamine-like and catechol0like, for their inhibitory effects on the uptake. The inhibitions were affected by changes in pH with changes in opposite directions found for the two series indicating the need for a positive charge in the side chain and suggesting an effect of the negative charge on the ring. These charge characteristics agreed with the pH profile observed in uptake. Consequently, the two groups with opposite charge characteristics in norepinephrine both appear to function in the uptake process.     

A comparison of the inhibitory potency of reversibly acting inhibitors of anion transport on chloride and sulfate movements across the human red cell membrane

The effects of a variety of chemically diverse, reversibly acting inhibitors have been measured on both Cl^? and SO₄^2? equilibrium exchange across the human red cell membrane. The measurements were carried out under the same conditions (pH 6.3, 8°C) and in the same medium for both the Cl^? and SO₂⁴ tracer fluxes. Under these conditions the rate constant for Cl^?-Cl^? exchange is about 20 000 times larger than that for SO₄^2?-SO₄^2? exchange. Despite this large difference in the rates of transport of the two anions, eight different reversibly acting inhibitors have virtually the same effect on the Cl^? and SO₄^2? transport. The proteolytic enzyme papain also has the same inhibitory effect on both the Cl^? and SO₄^2? self-exchange. In addition, the slowly penetrating disulfonate 2-(4′-aminophenyl)-6-methylbenzenethiazol-3′,7-disulfonic acid (APMB) is 5-fold more effective from the outer than from the inner membrane surface in inhibiting both Cl^? and SO₄^2? self-exchange. We interpret these results as evidence that the rapidly penetrating monovalent anion Cl^? and the slowly penetrating divalent anion SO₄^2? are transported by the same system.     

Cl−HCO3 exchange in rat renal basolateral membrane vesicles

Pathways for HCO₃⁻ transport across the basolateral membrane were investigated using membrane vesicles isolated from rat renal cortex. The presence of Cl⁻---HCO₃⁻ exchange was assessed directly by ³⁶Cl⁻ tracer flux measurements and indirectly by determinants of acridine orange absorbance changes. Under 10% CO₂/90% N₂ the imposition of an outwardly directed HCO₃⁻ concentration gradient (pH_o 6/pH_i 7.5) stimulated Cl⁻ uptake compared to Cl⁻ uptake under 100% N₂ in the presence of a pH gradient alone. Mediated exchange of Cl⁻ for HCO₃ was suggested by the HCO₃⁻ gradient-induced concentrative accumulation of intravesicular Cl⁻. Maneuvers designed to offset the development of ion-gradient-induced diffusion potentials had no significant effect on the magnitude of HCO₃⁻ gradient-driven Cl⁻ uptake further suggesting chemical as opposed to electrical Cl−HCO₃⁻ exchange coupling. Although basolateral membrane vesicle Cl⁻ uptake was observed to be voltage sensitive, the DIDS insensitivity of the Cl conductive pathway served to distinguish this mode of Cl⁻ translocation from HCO₃ gradient-driven Cl⁻ uptake. No evidence for cotransport was obtained. As determined by acridine orange absorbance measurements in the presence of an imposed pH gradient (pH_o 7.5/pH_i 6), a HCO₃⁻ dependent increase in the rate of intravesicular alkalinization was observed in response to an outwardly directed Cl⁻ concentration gradient. The basolateral membrane vesicle origin of the observed Cl⁻−HCO₃⁻ exchange activity was verified by experiments performed with purified brush-border membrane vesicles. In contrast to our previous observations of the effect of Cl⁻ on HCO₃⁻ gradient-driven Na⁺ uptake suggesting a basolateral membrane Na⁺−HCO₃⁻ for Cl⁻ exchange mechanism, no effect of Na⁺ on Cl−HCO₃⁻ exchange was observed in the present study.     

Investigation of the interaction of gold(III)-alkyldiamine complexes with l-histidine and imidazole ligands by H and C NMR, and UV spectrophotometry

Reactions of Au(III)-alkyldiamine complex with l-histidine and imidazole were carried out and monitored time-dependant by ¹H and ¹³C NMR. Kinetics for the [Au(en)Cl₂]⁺ reaction with l-histidine was determined by initial rate method at constant pH and 25 °C using UV-Vis absorption technique, and found to be first order with respect to each component, with a pseudo second order rate constant of 39 ± 3 M⁻¹ s⁻¹. Reaction rates of l-histidine and imidazole reactions with the [Au(en)Cl₂]⁺ complex was found to be strongly dependant on pD. The pD also has profound effect on the stability of the complex. It was observed that concurrent redox reactions also take place in solution in which Au(III) is reduced to metallic Au(0), while l-histidine and imidazole are oxidized to oxy and hydroxyl products. The optimization of the structure of [(His)Au(en)]³⁺ complex was carried out by gaussian03 at the RB3LYP level that showed a distorted square pyramid with the histidine carboxyl group at the pyramid top.     

13C nmr studies of aurothioglucose: Ligand exchange and redox disproportionation reactions

¹³C nmr studies of gold thioglucose, AuSTg, and solutions containing added β-1-D-thioglucose, TgSH, have been conducted at PD 7.4 and interpreted in terms of complexation and ligand exchange reactions that are consistent with the known preference of gold(I) for linear two-coordinate structures. The upper limit of the half-life for ligand exchange between 0.25 M Au(STg)₂^? and TgSH at pD 7.4 is 2.2 msec. The ¹³C nmr spectra of various thioglucose derivatives have been assigned. A novel oxidation reduction reaction was discovered that leads to the formation of metallic gold and a product tentatively identified as the sulfinic acid derivative of thioglucose. The presence of sulfinic acid in AuSTg was indicated by the infrared absorption at 1050 cm^?1. The same product was formed by slow hydrolysis of thioglucose disulfide. A mechanism for the formation of the sulfinic acid derivative from AuSTg is proposed.     

Chemosensory responses of Tetrahymena thermophila to CB2, a 24-amino-acid fragment of lysozyme

While lysozyme is a depolarizing chemorepellent in Tetrahymena, the entire lysozyme molecule is not necessary to activate the lysozyme receptor. Reduced lysozyme was cut into three fragments by cyanogen bromide cleavage and the fragments (CB₁, CB₂ and CB₃) were separated by HPLC. Behavioral bioassays showed that the carboxy-terminal 24-amino-acid fragment, which we call CB₂, is 100 times more active than intact lysozyme as a chemorepellent. CB₂ appears to activate the same receptor as lysozyme because behavioral cross-adaptation is seen between these two compounds and an antibody generated to the purified lysozyme receptor blocks responses to both lysozyme and CB₂. This is further supported by the observation that neomycin, which is a competitive inhibitor of lysozyme binding, also inhibits CB₂ responses. This inhibition may be due to the fact that neomycin is highly positively charged (+5 at pH 7.0) and CB₂ has a net charge of +4 at pH 7.0. Intracellular electrophysiological recordings documented that CB₂ elicits a transient, depolarizing receptor potential that is similar to the lysozyme-induced depolarizations except they are much smaller. CB₂ is a more potent and specific ligand for use in studies of the lysozyme receptor of Tetrahymena. Accepted: 21 February 1999     

Vibrational Raman optical activity of alanyl peptide oligomers: A new perspective on aqueous solution conformation

The vibrational Raman optical activity (ROA) spectra of di- and tri-L -alanine in the range 650–1750 cm^?1 have been measured in H₂O and D₂O solution at high, neutral, and low pH and pD. Corresponding ROA spectra for tetra- and penta-L -alanine have also been obtained, but over a more restricted set of pH and pD conditions. There are similarities with the ROA spectrum of L -alanine below ～ 1200 cm^?1, but the spectra are very different above this wavenumber due to the influence of the vibrational coordinates of the peptide group. The similar overall appearance of the di-, tri-, and tetrapeptide ROA under selected conditions of pH and pD, and of all four peptide ROA spectra in DCl and HCl solutions, in the backbone skeletal stretch region ～ 1050–1200 cm^?1 and the extended amide III region ～ 1250–1350 cm^?1, suggests that the backbone conformation is approximately the same in all four structures. One difference, however, is a shift of a large positive ROA band in H₂O at ～ 1341 cm^?1 in the dipeptide, assigned to C_α–H and in-plane N–H deformations, down to ～ 1331 cm ^?1 in the tripeptide and to ～ 1315 cm^?1 in the tetrapeptide and pentapeptide (the last in HCl due to insufficient solubility in H₂O), which indicates increasing delocalization of the corresponding normal mode with increasing chain length. Our results do not support the suggestion that stabilizing interactions of the zwitterionic end groups in tri-L -alanine at neutral pH leads to a different solution structure to that at high pH. © 1994 John Wiley & Sons, Inc.     

Protein charge ladders reveal that the net charge of ALS-linked superoxide dismutase can be different in sign and magnitude from predicted values

This article utilized “protein charge ladders”—chemical derivatives of proteins with similar structure, but systematically altered net charge—to quantify how missense mutations that cause amyotrophic lateral sclerosis (ALS) affect the net negative charge (Z) of superoxide dismutase-1 (SOD1) as a function of subcellular pH and Zn²⁺ stoichiometry. Capillary electrophoresis revealed that the net charge of ALS-variant SOD1 can be different in sign and in magnitude—by up to 7.4 units per dimer at lysosomal pH—than values predicted from standard pK_a values of amino acids and formal oxidation states of metal ions. At pH 7.4, the G85R, D90A, and G93R substitutions diminished the net negative charge of dimeric SOD1 by up to +2.29 units more than predicted; E100K lowered net charge by less than predicted. The binding of a single Zn²⁺ to mutant SOD1 lowered its net charge by an additional +2.33 ± 0.01 to +3.18 ± 0.02 units, however, each protein regulated net charge when binding a second, third, or fourth Zn²⁺ (ΔZ < 0.44 ± 0.07 per additional Zn²⁺). Both metalated and apo-SOD1 regulated net charge across subcellular pH, without inverting from negative to positive at the theoretical pI. Differential scanning calorimetry, hydrogen-deuterium exchange, and inductively coupled plasma mass spectrometry confirmed that the structure, stability, and metal content of mutant proteins were not significantly affected by lysine acetylation. Measured values of net charge should be used when correlating the biophysical properties of a specific ALS-variant SOD1 protein with its observed aggregation propensity or clinical phenotype.     

Structure and dynamics of the neutrophil defensins NP-2, NP-5, and HNP-1: NMR studies of amide hydrogen exchange kinetics

The exchange kinetics for the slowly exchanging amide hydrogens in three defensins, rabbit NP-2, rabbit NP-5, and human HNP-1, have been measured over a range of pH at 25°C using 1D and 2D NMR methods. These NHs have exchange rates 10² to 10⁵ times slower than rates from unstructured model peptides. The observed distribution of exchange rates under these conditions can be rationalized by intramolecular hydrogen bonding of the individual NHs, solvent accessibility of the NHs, and local fluctuations in structure. The temperature dependencies of NH chemical shifts (NH temperature coefficients) were measured for the defensins and these values are consistent with the defensin structure. A comparison is made between NH exchange kinetics, NH solvent accessibility, and NH temperature coefficients of the defensins and other globular proteins. Titration of the histidine side chain in NP-2 was examined and the results are mapped to the three-dimensional structure. © 1994 Wiley-Liss, Inc.     

Secretion of HCO 3 − /OH− in cortical distal tubule of the rat

Secretion of bicarbonate has been described for distal nephron epithelium and attributed to apical Cl^–/HCO ₃ ^– exchange in beta-intercalated cells. We investigated the presence of this mechanism in cortical distal tubules by perfusing these segments with acid (pH 6) 10 mm phosphate Ringer. The kinetics of luminal alkalinization was studied in stationary microperfusion experiments by double-barreled pH (ion-exchange resin)/1 m KCl reference microelectrodes. Luminal alkalinization may be due to influx (into the lumen) of HCO ₃ ^– or OH^–, or efflux of H⁺. The magnitude of the Cl^–/ HCO ₃ ^– exchange component was measured by perfusing the lumen with solutions with or without chloride, which was substituted by gluconate. This component was not different from zero in control and alkalotic (chronic plus acute) Wistar rats. Homozygous Brattleboro rats (BRB), genetically devoid of antidiuretic hormone, were used since this hormone has been shown to stimulate H⁺ secretion, which could mask bicarbonate secretion. In these rats, no evidence for Cl^–/HCO ₃ ^– exchange was found in control BRB and in early distal segments of alkalotic animals, but in late distal tubule a significant component of 0.14±0.033 nmol/cm² · sec was observed, which, however, is small when compared to the reabsorptive flow found in control Wistar rats, of 0.95±0.10 nmol/cm² · sec. In addition, 5×10^–4 m SITS had no effect on distal bicarbonate reabsorption in controls as well as on secretion in alkalotic Wistar and Brattleboro rats, which is compatible with the absence of effect of this drug on the apical Cl^–/HCO ₃ ^– exchange in other tissues. It is concluded that most distal alkalinization is not Cl^– dependent, and that Cl^–/HCO ₃ ^– exchange may be found in cortical distal tubule, but its magnitude is, even in alkalosis, markedly smaller than the reabsorptive flux, which predominates in the rats studied in this paper, keeping luminal pH lower than that of blood.