Remeasuring HEWL pK(a) values by NMR spectroscopy: methods, analysis, accuracy, and implications for theoretical pK(a) calculations

Site-specific pK(a) values measured by NMR spectroscopy provide essential information on protein electrostatics, the pH-dependence of protein structure, dynamics and function, and constitute an important benchmark for protein pK(a) calculation algorithms. Titration curves can be measured by tracking the NMR chemical shifts of several reporter nuclei versus sample pH. However, careful analysis of these curves is needed to extract residue-specific pK(a) values since pH-dependent chemical shift changes can arise from many sources, including through-bond inductive effects, through-space electric field effects, and conformational changes. We have re-measured titration curves for all carboxylates and His 15 in Hen Egg White Lysozyme (HEWL) by recording the pH-dependent chemical shifts of all backbone amide nitrogens and protons, Asp/Glu side chain protons and carboxyl carbons, and imidazole protonated carbons and protons in this protein. We extracted pK(a) values from the resulting titration curves using standard fitting methods, and compared these values to each other, and with those measured previously by 1H NMR (Bartik et al., Biophys J 1994;66:1180–1184). This analysis gives insights into the true accuracy associated with experimentally measured pK(a) values. We find that apparent pK(a) values frequently differ by 0.5–1.0 units depending upon the nuclei monitored, and that larger differences occasionally can be observed. The variation in measured pK(a) values, which reflects the difficulty in fitting and assigning pH-dependent chemical shifts to specific ionization equilibria, has significant implications for the experimental procedures used for measuring protein pK(a) values, for the benchmarking of protein pK(a) calculation algorithms, and for the understanding of protein electrostatics in general.     

Ionization behavior of acidic residues in calbindin D(9k).

The ionization state of seven glutamate residues, one aspartate, and the C-terminal alpha-COOH group in bovine apo calbindin D(9k) has been studied by measurement and modeling of the pH titration curves and apparent pK(a) values. The observed pK(a) ranged from 3.0 to 6.5. Most of the observed acidic groups were half-ionized at lower pH values than those in unstructured proteins. As a rule, the ionization equilibria extended over a wider pH range than in the case of unperturbed single titrations, indicating a complex influence of protein charges on the charge state of each individual residue. Glu17, which is a backbone Ca(2+)-ligand in the N-terminal binding loop of calbindin D(9k), was half-protonated at pH 3.6 but manifested biphasic titration with apparent pK(a) values of 3.2 and 6.5. Complementary Monte Carlo simulations of the titration process and pK(a) values of the acidic groups in calbindin D(9k) reproduce the experimentally observed titration features, except for the pronounced double titration of Glu17. Discrepancies between the results from direct measurement and from modeling may be partly caused by changes in the protein structure when the net charge changes from -8 to +11 over the isoelectric point at pH 5. Proteins 1999;37:106-115.     

Mathematical modelling of charge properties of protein molecules]

A procedure of theoretical determination of the dependence of protein molecule charge on the medium pH has been developed. The suggested procedure allows calculating the protein pI value, the molecule charge at the definite pH value, as well as the corresponding values for the protein molecule. Calculations for insulin, apo A-I and apo A-II molecules have been carried out. Calculated pI values are equal to 5.25, 5.64 and 4.86, respectively. A comparison of the theoretical curves and experimental data allows obtaining information of the molecule structure. Carboxyl groups with abnormally high pK values are discovered, that, probably, indicates to the direct interaction of two COOH-groups. A supposition is made on the most probable arrangement of the functional fragments in apo A-I and apo A-II molecules.     

Kinetic study of the pH-dependence of maximal rate of Ca-ATP hydrolysis by myosin]

Curves of V pH-dependence for Ca ATPase of myosin and heavy meromyosin are demonstrated to be well modelled with theoretical curves for the case of proton dissociation at three groups of enzyme-substrate complex with the loss of the activity at some intermediate ionization stage. Variation of pK values for these three groups and the degree of inhibition for intermediate forms of enzyme-substrate complex are found to be sufficient to reproduce main varieties of described in the literature and obtained in this work multiformity of pH-dependence curves of different nucleoside triphosphates hydrolysis by both native and modified enzymes. Calculated pK values and modification data suggest a significant importance of the dissociation of two imidazole groups ("activating" and "inhibitory") and cisteine sulhydryl group for the catalytic activity of myosin. Inhibition of ATPase activity by increasing of KCl concentrations is found to be due first of all to a shift in pK values of "inhibitory" imidazole and sulhydryl groups.     

Electrostatic effects in hemoglobin: hydrogen ion equilibria in human deoxy- and oxyhemoglobin A.

The modified Tanford-Kirkwood theory of Shire et al. [Shire, S. J., Hanania, G.I.H., & Gurd, F.R.N. (1974) Biochemistry 13, 2967] for electrostatic interactions was applied to the hydrogen ion equilibria of human deoxyhemoglobin and oxyhemoglobin. Atomic coordinates for oxyhemoglobin were generated by the application of the appropriate rigid rotation function to alpha and beta chains of the deoxyhemoglobin structure [Fermi, G. (1975) J. Mol. Biol. 97, 237]. The model employs two sets of parameters derived from the crystalline protein structures, the atomic coordinates of charged amino acid residues and static solvent accessibility factors to reflect their individual degrees of exposure to solvent. Theoretical titration curves based on a consistent set of pKint values compared closely with experimental potentiometric curves. Theoretical pK values at half-titration for individual protein sites corresponded to available observed values for both quaternary states. The results bring out the cumulative effects of numerous electrostatic interactions in the tetrameric structures and the major effects of the quaternary transition that result from changes in static solvent accessibility of certain ionizable groups.     

The uptake of protons by heme-linked ionizable groups on azide binding to methemoglobin

When azide ion reacts with methemoglobin in unbuffered solution the pH of the solution increases. This phenomenon is associated with increases in the pK values of heme-linked ionizable groups on the protein which give rise to an uptake of protons from solution. We have determined as a functional of pH the proton uptake, delta h+, on azide binding to methemoglobin at 20 degrees C. Data for methemoglobins A (human), guinea pig and pigeon are fitted to a theoretical expression based on the electrostatic effect of these sets of heme-linked ionizable groups on the binding of the ligand. From these fits the pK values of heme-linked ionizable groups are obtained for liganded and unliganded methemoglobins. In unliganded methemoglobin pK1, which is associated with carboxylic acid groups, ranges between 4.0 and 5.5 for the three methemoglobins; pK2, which is associated with histidines and terminal amino groups, ranges from 6.2 to 6.7. In liganded methemoglobin pK1 lies between 5.8 and 6.3 and pK2 varies from 8.1 to 8.5. The pH dependences of the apparent equilibrium constants for azide binding to the three methemoglobins at 20 degrees C are well accounted for with the pK values calculated from the variation of delta h+ with pH.     

Competitive labelling, a method for determining the reactivity of individual groups in proteins. The amino groups of porcine elastase

1. A method is described for determining the ionization constants and reactivities of individual amino groups in proteins. The principle is that in the presence of a trace amount of radioactive label, the various reactive groups in a protein molecule will compete for the label and the amount incorporated into any one group will be determined by its nucleophilicity, pK and micro-environment. The relative amounts of label incorporated into various groups will be proportional to their second-order rate constants and by comparing these rate constants with those expected on the basis of a linear free-energy relationship obtained with a series of standard compounds, the micro-environment can be defined for a particular amino group. 2. The method consists of treating a protein and an internal standard with a limiting amount of radioactive reagent and then with an excess of unlabelled reagent to yield a chemically homogeneous but heterogeneously labelled compound. After appropriate enzymic digestion peptides containing each labelled group are isolated and their rates of reaction, relative to the internal standard, are determined from their specific radioactivities. The entire procedure is repeated at several pH values. 3. When the method was applied to the amino groups of porcine elastase by using tritiated acetic anhydride as the labelling reagent, the N-terminus was found to have pK(a) 9.7 and a much lower than normal reactivity. Lysine-87 and lysine-224 were found to have pK(a) 10.3 and normal reactivities. At pH values greater than 10.5 there are discontinuities in all the titration curves, indicating that the entire molecule is undergoing a structural reorganization.     

Curves of ligand binding. The use of hyperbolic functions for expressing titration curves

1. The dependences of the concentrations of the non-ligated, uni-ligated and bi-ligated forms of a molecule that binds two molecules of ligand are expressed as functions of the logarithm of free ligand concentration by means of hyperbolic functions. Expressions are also given for the saturation both of an individual site and of the molecule as a whole. This form of expression allows derivation of the following points. 2. The sharpness of bell-shaped curves of concentration of the uni-ligated form is analysed in terms of the heights of their points of inflexion; these can rise to 1/ radical2 of the curve. 3. A single group can exhibit a doubly sigmoid saturation curve if this group and another have comparable affinities for a ligand, and if ligand binding at one of them diminishes the affinity at the other. If the molecular pK values pK(1) and pK(2) for the first and second molecules of ligand are called pK*+/-logm, so that K*(2)=K(1)K(2) and m(2)=K(1)/K(2), then the doubly sigmoid curve can be represented by the sum of two independent one-site saturation curves, in general of unequal height, of pK values pK*+/-log(1/2)[m+ radical(m(2)-4)]. The error in such representation is small either if the mutual interaction between the groups (i.e. m) is large, or if the groups have very similar affinities for the ligand. 4. The sum of two one-site saturation curves, again of pK values of pK*+/-log(1/2)[m+ radical(m(2)-4)] but of equal heights, gives a precise value for the total saturation, provided that the binding of one molecule does not promote the binding of a second, i.e. providing that m>/=2. Hence determinations of saturation cannot distinguish interacting and possibly identical sites from independent and different ones.     

Effect of pH on inhibition and spontaneous reactivation of acetylcholinesterase treated with esters of phosphorus acids and of carbamic acids

1. The second-order rate constants of inhibition, k(a), of acetylcholinesterase were measured at pH values between 5.5 and 10.5 for two esters of phosphorus acids and five esters of carbamic acids. Two of the carbamates and one of the phosphates contained a quaternary nitrogen group. 2. For the three positively charged compounds the k(a)-pH plots are bell-shaped, with a pH optimum between 7.5 and 9.0. The changes in k(a) above and below the optimum pH fit theoretical curves for the dissociation of groups on the protein of pK 6.2 and 10.25. 3. For the uncharged compounds, the k(a)-pH plot on the alkaline side is identical with the one obtained for charged inhibitors. On the acid side they do not fit such a curve and the k(a) for two of the carbamates is independent of pH changes between 5.5 and 8.0. 4. The first-order rate constants, k(+3), for spontaneous reactivation were measured at pH values between 5.0 and 11.0 for N-methylcarbamoylated, NN-dimethylcarbamoylated and di-(2-chloroeth)phosphorylated cholinesterase. For all three derivatives the k(+3)-pH plots are bell-shaped, with a pH optimum between 8.0 and 8.5. The changes in k(+3) above and below the optimum fit theoretical curves for the dissociation of groups of pK 6.9 and 9.8. 5. The relevance of these results to binding, acylation and deacylation of both inhibitors and substrates is discussed.     

Progress in the prediction of pKa values in proteins

The pK(a) -cooperative aims to provide a forum for experimental and theoretical researchers interested in protein pK(a) values and protein electrostatics in general. The first round of the pK(a) -cooperative, which challenged computational labs to carry out blind predictions against pK(a) s experimentally determined in the laboratory of Bertrand Garcia-Moreno, was completed and results discussed at the Telluride meeting (July 6-10, 2009). This article serves as an introduction to the reports submitted by the blind prediction participants that will be published in a special issue of PROTEINS: Structure, Function and Bioinformatics. Here, we briefly outline existing approaches for pK(a) calculations, emphasizing methods that were used by the participants in calculating the blind pK(a) values in the first round of the cooperative. We then point out some of the difficulties encountered by the participating groups in making their blind predictions, and finally try to provide some insights for future developments aimed at improving the accuracy of pK(a) calculations.     

Theoretical calculation of pKa reveals an important role of Arg205 in the activity and stability of Streptomyces sp. N174 chitosanase

Based on the crystal structure of chitosanase from Streptomyces sp. N174, we have calculated theoretical pK(a) values of the ionizable groups of this protein using a combination of the boundary element method and continuum electrostatics. The pK(a) value obtained for Arg(205), which is located in the catalytic cleft, was abnormally high (>20.0), indicating that the guanidyl group may interact strongly with nearby charges. Chitosanases possessing mutations in this position (R205A, R205H, and R205Y), produced by Streptomyces lividans expression system, were found to have less than 0.3% of the activity of the wild type enzyme and to possess thermal stabilities 4-5 kcal/mol lower than that of the wild type protein. In the crystal structure, the Arg(205) side chain is in close proximity to the Asp(145) side chain (theoretical pK(a), -1.6), which is in turn close to the Arg(190) side chain (theoretical pK(a), 17.7). These theoretical pK(a) values are abnormal, suggesting that both of these residues may participate in the Arg(205) interaction network. Activity and stability experiments using Asp(145)- and Arg(190)-mutated chitosanases (D145A and R190A) provide experimental data supporting the hypothesis derived from the theoretical pK(a) data and prompt the conclusion that Arg(205) forms a strong interaction network with Asp(145) and Arg(190) that stabilizes the catalytic cleft.     

Proton-magnetic-resonance studies of the lysine residues of ribonuclease A.

The amino groups of ribonuclease A (RNase-A) have been methylated with formaldehyde and borohydride to provide observable resonances for proton magnetic resonance (PMR) studies. Although enzymatic activity is lost, PMR difference spectroscopy and PMR studies of thermal denaturation show native conformation is largely preserved in methylated RNase-A. Resonances corresponding to the NH2-terminal alpha-amino and 10 xi-amino N-methyl groups are titrated at 220 MHz to obtain pK values. After correction for the effects of methylation, using values previously derived from model compound studies, a pK of 6.6 is found for the alpha-amino group, a pK of 8.6 for the xi-amino group of lysine-41 and pK values ranging from 10.6 to 11.2 for the other lysine xi-amino groups. Interactions between lysine-7 and lysine-41 or between the alpha-amino and xi-amino groups of lysine-1 have been proposed to account for deviations from simple titration behaviour. The correct continuities for the titration curves of the histidine H-2 proton resonances have been confirmed by selective deuteration of the H-2 protons. Titration curves for the H-2 proton resonances of histidine-12 and histidine-119 of methylated RNase-A show deviations from the titration curves for the native enzyme, indicating some alteration of the active-site conformation. In the presence of phosphate, titration curves for the H-2 proton resonances of histidine-12 and histidine-119 of methylated RNase-A indicate binding of phosphate at the active site, but these curves continue to show deviations from the titration behaviour of native RNase-A. The titration curve for the N-methyl resonance of lysine-41 is perturbed considerably by the presence of phosphate, which indicates a possible catalytic role for lysine-41.     

Empirical relationships between protein structure and carboxyl pKa values in proteins

Relationships between protein structure and ionization of carboxyl groups were investigated in 24 proteins of known structure and for which 115 aspartate and 97 glutamate pK(a) values are known. Mean pK(a) values for aspartates and glutamates are < or = 3.4 (+/-1.0) and 4.1 (+/-0.8), respectively. For aspartates, mean pK(a) values are 3.9 (+/-1.0) and 3.1 (+/-0.9) in acidic (pI < 5) and basic (pI > 8) proteins, respectively, while mean pK(a) values for glutamates are approximately 4.2 for acidic and basic proteins. Burial of carboxyl groups leads to dispersion in pK(a) values: pK(a) values for solvent-exposed groups show narrow distributions while values for buried groups range from < 2 to 6.7. Calculated electrostatic potentials at the carboxyl groups show modest correlations with experimental pK(a) values and these correlations are not improved by including simple surface-area-based terms to account for the effects of desolvation. Mean aspartate pK(a) values decrease with increasing numbers of hydrogen bonds but this is not observed at glutamates. Only 10 pK(a) values are > 5.5 and most are found in active sites or ligand-binding sites. These carboxyl groups are buried and usually accept no more than one hydrogen bond. Aspartates and glutamates at the N-termini of helices have mean pK(a) values of 2.8 (+/-0.5) and 3.4 (+/-0.6), respectively, about 0.6 units less than the overall mean values.     

Ionization and reactivities of the thiol groups which participate in the formation of interchain disulfide bonds of Bence Jones proteins and an Fab(t) fragment

The pK values and reactivities of the thiol groups which participate in the formation of interchain disulfide bonds in Bence Jones proteins and the Fab(t) fragment of a myeloma protein (Jo) (IgGl, kappa) were determined by means of the reactions with chloroacetamide and DTNB, and of spectrophotometric titration. The two thiol groups of partially reduced type kappa Bence Jones protein dimers had the same pK values (pK = 9.76 at 0.2 ionic strength and 25 degrees C) and the same true second-order rate constants (k) toward chloroacetamide (k = 18.8 x 10(-2) M-1 . S-1). The two thiol groups of partially reduced type lambda Bence Jones protein dimers had different pK values but the variation of the pK values among the specimens was small (pK1 = 8.5-8.6 and pK2 = 9.5-9.7 at 0.2 ionic strength and 25 degrees C). The spectrophotometric titration of partially reduced Nag protein (type lambda) also showed that the two thiol groups have different pK values. The pK values of two thiol groups of the partially reduced Fab(t) fragment were determined as 8.51 and 9.76 at 0.2 ionic strength and 25 degrees C. The effect of ionic strength on the pK values of the thiol groups of partially reduced Nag protein and the pK values of the thiol groups in partially reduced Ta protein (type kappa) and in a hybrid molecule formed between partially reduced Ta protein and partially reduced and alkylated H chains indicated that the difference in pK values did not arise from electrostatic interaction between the two thiol groups, but that the pK values are intrinsically different. The true rate constants, k1 and k2, of the two thiol groups of type lambda Bence Jones proteins varied with the specimen (k1 = 1.9-5.7 x 10(-2) M-1 . S-1 and k2 = 18.5-25.0 x 10(-2) M-1 . S-1). The k1 and k2 values for Jo-Fab(t) were 7.21 x 10(-2) and 23.1 x 10(-2) M-1 . S-1, respectively. On the basis of these pK values and reactivities, we discuss the reformation of the interchain disulfide bonds from partially reduced Bence Jones proteins and immunoglobulins in the presence of oxidized glutathione.     

1H-NMR study on the tautomerism of the imidazole ring of histidine residues. I. Microscopic pK values and molar ratios of tautomers in histidine-containing peptides

The 1H-NMR titration curves of chemical shifts versus pH were observed for imidazole, N1-methylimidazole, L-histidine, N1-methyl-L-histidine, N3-methyl-L-histidine, and other related compounds. With these results, the macroscopic pK values of these compounds were determined by a computer curve-fitting for a simple dissociation sequence. From the pK values of imidazole and N1-methylimidazole, the perturbation for the pK of the imidazole ring due to the substitution of a proton with a methyl group was estimated as -0.21 pH unit. The microscopic pK values of the individual tautomers of the imidazole ring were estimated with the pK values of N1-methyl-L-histidine, N3-methyl-L-histidine, and perturbation due to methyl substitution. The estimated pK values were 6.73 for the N1-H tautomer and 6.12 for the N3-H tautomer. These values were in good agreement with those obtained using carboxymethyl derivatives instead of methyl derivatives. Furthermore, the macroscopic pK value (6.02) calculated using the estimated microscopic pK values agreed with that (6.03) observed for the imidazole ring of L-histidine. Thus the method in this work was indicated to be self-consistent. The microscopic pK values of tautomers were also obtained for N alpha-acetyl-L-histidine and N alpha-acetyl-L-histidine methylamide. The molar ratios of tautomers were calculated on the basis of the microscopic pK values of tautomers. The intrinsic (or unperturbed) pK value of imidazole ring and perturbations due to the CO2- and NH3+ were obtained for each of the N1-H and N3-H tautomers.     

Effect of pH on the Kinetic Parameters of NADP-Malic Enzyme from a C(4)Flaveria (Asteraceae) Species

We have used the pH variation in the kinetic parameters with respect to malate of NADP-malic enzyme purified from the C₄ species, Flaveria trinervia, to compare the pK values of its functional groups with those for the pigeon liver NADP-malic enzyme (MI Schimerlik, WW Cleland [1977] Biochemistry 16: 576-583) and the plant NAD-malic enzyme (KO Willeford, RT Wedding [1987] Plant Physiol 84: 1084-1087). Like the other enzymes, the C₄ enzyme has a group with a pK of about 6.0 (6.6 for the C₄ enzyme), as indicated from plots of the log V_max/K_m (V_max = maximum rate of catalysis) versus pH, which must lose a proton for malate binding and subsequent catalysis. The optimum ionization for the C₄ enzyme-NADP-Mg²⁺ complex occurs at pH 7.1 to 7.5. From pH 7.5 to 8.4, the K_m increases, but V_max remains constant. The log V_max/K_m plot in this pH range indicates a group with a pK of about 7.7. The other malic enzymes exhibit a similar pK. Above pH 8.4, deprotonation leads to a marked increase in K_m and a decrease in V_max for the C₄ enzyme. As in the case of the animal enzyme, the log V_max/K_m plot for the C₄ enzyme appears to approach a slope of two. The curve suggests an average pK of 8.4 for the groups involved, while the animal enzyme exhibits an average pK of 9.0. The NAD-malic enzyme does not exhibit any pK values at these high pK values. We hypothesize that the putative groups with the high pK values may be at least partially responsible for the ability of the C₄ NADP-malic enzyme to maintain high activity at pH 8.0 in illuminated chloroplasts.     

pK(a) calculations of calbindin D(9k): effects of Ca(2+) binding, protein dielectric constant, and ionic strength

Calbindin is a small (75 residues) helix-loop-helix ("EF-hand") calcium-binding protein belonging to the calmodulin superfamily. It binds two Ca(2+) ions. Continuum electrostatics in combination with the boundary element method was employed for the calculation of the acid-dissociation constants K(a) (pK(a) = -log K(a)) values of all titratable residues in the protein. The objectives were to determine quantitatively the effects of divalent ion binding and small ion-induced structural changes on predicted pK(a)'s. Computations were carried out for the apo and holo form of calbindin, for which both X-ray and NMR structures were available. Comparison was made with several sets of experimental pK(a) values determined by NMR spectroscopy. Different choices of the dielectric constant (ranging from 4 to 78.5) for calbindin and variations in ionic strength (from 0 to 0.3 M) were investigated in a systematic fashion. Removal of the two bound Ca(2+) ions increases the pK(a) values of all residues if no conformational changes were allowed. If conformational differences between the apo and holo were accounted for, shifts in either direction were observed. Titrating groups that are directly involved in Ca(2+) binding (Asp and Glu) required a dielectric constant of 78.5 for the holo structure to obtain a reasonable estimate of their pK(a)'s. For the apo structure, passable values for the pK(a)'s of these ligating groups could be determined if the structure was allowed to relax upon ion removal.     

Titration of E. coli transhydrogenase domain III with bound NADP+ or NADPH studied by NMR reveals no pH-dependent conformational change in the physiological pH range

A pH-titration 2D NMR study of Escherichia coli transhydrogenase domain III with bound NADP(+) or NADPH has been carried out, in which the pH was varied between 5.4 and 12. In this analysis, individual amide protons served as reporter groups. The apparent pK(a) values of the amide protons, determined from the pH-dependent chemical shift changes, were attributed to actual pK(a) values for several titrating residues in the protein. The essential Asp392 is shown to be protonated at neutral pH in both the NADP(+) and NADPH forms of domain III, but with a marked difference in pK(a) not only attributable to the charge difference between the substrates. Titrating residues found in loop D/alpha5 point to a conformational difference of these structural elements that is redox-dependent, but not pH dependent. The observed apparent pK(a) values of these residues are discussed in relation to the crystal structure of Rhodospirillum rubrum domain III, the solution structure of E. coli domain III and the mechanism of intact proton-translocating transhydrogenase.     

Empirical parametrization of pK values for carboxylic acids in proteins using a genetic algorithm

Considerable effort has been devoted to the development of theoretical electrostatic methods to predict the pK values of ionizable residues in proteins. However, predictions appear often to be still at the qualitative or semi-quantitative level. We believe that, with the increasing number experimentally available pK values for proteins of known structure, an alternative approach becomes feasible: the empirical parametrization of the experimental protein pK database. Of course, in the long term, this empirical approach is no substitute for rigorous electrostatic analysis but, in the short term, it may prove to have useful predictive power and it may help to pinpoint the main structural determinants of pK values in proteins. Here we demonstrate the feasibility of the parametrization approach by fitting (using a genetic algorithm as fitting tool) the database for carboxylic acid pK values in proteins on the basis of an empirical equation that takes into account the two following kinds of effects: (1) long-range charge-charge interactions; (2) interactions of the given carboxylic acid group with its environment in the protein, which are described in terms of contributions from the different kind of atoms present in the protein (atomic contributions).     

Studies on groundnut proteins. VI. Isolation, characterisation and hydrogen ion titration of conarachin II.

A method is described for isolating conarachin II in a homogeneous form by the techniques of DEAE-cellulose chromatography, polyacrylamide gel electrophoresis and sedimentation velocity. The protein contains 0.72% carbohydrate and no phosphate. Hydrogen ion titration curve indicated that the sidechain carboxyl, imidazol and epsilon-amino groups titrated with normal pK Int values and their number agreed with the analytical values obtained from amino acid analysis. However, tyrosine phenolic groups had abnormal pK Int of 10.5.