pK Of Group Research Articles

Adsorption on packed bed electrodes

The potential-dependent sorption which occurs on a graphite surface is quite different from that on metals and permits a wide range of materials from molecules to bacteria to be sorbed. The active sites for this process are the edges of the graphite layer planes where the pK of the surface functional groups is modified by the applied potential to control the sorption in a manner analogous to that on an ion exchange resin.

Proton Overhauser experiments on kringle 4 from human plasminogen. Implications for the structure of the kringles' hydrophobic core

1H-NMR Overhauser experiments at 300 and 600 MHz have been implemented on the isolated kringle 4 fragment of human plasminogen. This study shows that Leu 46 and Leu 77 CH 3 δ,δ′ groups, as well as two threonine CH 3 γ and a methionine S-CH ϵ (probably Met 48 groups, are in efficient dipolar contact with histidine and aromatic side-chains. In particular, the experiments reveal that of the two Leu 46 CH 3 δ,δ′ groups, one is in efficient contact with tryptophan (Trp 25 and Trp 62 indole rings while the other interacts with a tyrosine (probably Tyr 41) phenol. Leu 46 appears also to be close to an Ala CH 3 β group. Such a hydrophobic cluster appears to be contiguous to Trp 72, hence to Arg 71, residues that are through to be part of the lysine-binding site. Acid-base titration experiments show that the buried methionine S-CH 3 ϵ group senses a neighboring ionizable group pK ∗ 1 = 3.76 , suggesting presence of a carboxyl anionic group (probably an aspartic acid side-chain) in the vicinity of the hydrophobic core. A preliminary model is proposed for the overall folding of the kringle polypeptide chain.

Fluorescence properties of some isoquinoline alkaloids

The fluorescence properties of 28 isoquinoline alkaloids have been investigated. In most of them the chromophore responsible for fluorescence was the benzene ring with electron-donor substituents containing oxygen. The long-wave excitation peak practically coincides with the long-wave absorption peak of these substances, covering the region from 284 to 293 nm, the maximum emission being in the range 320-332 nm. With alkaloids having a number of conjugated rings, both excitation and emission were observed at higher wavelengths. Only protoberberine alkaloids behaved as hydrophobic probes, i.e. transfer of these compounds into a less polar medium produced a marked hypsochromic shift and a higher intensity of emission. The effect of polarity on the behaviour of tetrahydroprotoberberines, protopines, pavinanes, aporphines and benzophenanthridines was not so pronounced. Changes of pH manifested themselves most markedly in compounds with dissociable hydroxyl groups; the majority of phenolates did not fluoresce.The phenol group pK values of these compounds in the excited state were lower than in the ground state (which ranged between 8.4 and 10.4). The relations between the apparent pK, determined from fluorescence data, and the pK's of these compounds in the ground and the excited states are discussed.

Pulse radiolysis studies of WR-1065

WR-1065, the dephosphorylated sulfhydryl form of WR-2721 has been suggested as the metabolite responsible for the radioprotective abilities of the latter and as being active during the radiation chemical stage of damage production. Hence this study was performed to determine some of the radiation chemical parameters of the compound. Pulse radiolysis techniques, developed for use with SH compounds, were employed in the current work: Three systems were used to attempt to measure the rate constant for reaction of OH' radicals with WR-1065; (a) Competition with phenylalanine, in which the decrease in the amount of RSSR- caused by the addition of phenylalanine is monitored; this failed because of the absence of an absorbance attributable to RSSR-. (b) Competition with 2,2′-Azinobis-(3-ethylbenzthiazo6ne-6-sulfonate) (ABTS~ monitoring the decrease in OH' induced ABTS absorbance caused by WR-1065 addition. This was unsuccessful due to reaction of RS with ABTS. (c) Competition with CNS −, observing the decrease in (CNS) 2 −caused by WR-1065 addition indicates that the second order rate constant for reaction of OH' with WR-1065 is 9.2 ± 03 X 109 M −1 s −1 To investigate the ability of WR-1065 to react with DNA radicals the yield of ABTS radicals was monitored in a situation where DNA scavenges 50% of the OH' radicals in the presence of ABTS (20°6) and WR-1065 (3096). DNA radicals were shown not to react with ABTS but WR-1065 radicals do, the absence of additional ABTS absorbing species indicates that DNA radicals do not react with WR-1065 under the experimental conditions used. An attempt was made to investigate the possibility that WR-1065 is concentrated close to DNA macromolecules in solution: The rate constant for reaction Br 2 staggered− with WR-1065 was measured in the presence and in the absence of excess DNA (with which Br 2 staggered− was not observed to react). The observed rates of reaction were independent of the presence of DNA indicating that the latter does not affect the availability of WR-1065 for reaction with Br 2 staggered− radical. Reaction of WR-1065 with Br 2 − was used as a probe for determining the pK of the SH group. The rate of reaction increases from 1.8 × 10 8 M −1 s −1 at pH 6.3 to 1.6 × 109 M −1 s −1 at pH 8.4, the mid-point of the increase indicates a pK of 7.3.

Polarography of n-butylthioglycolate in aqueous media, methanol, and acetonitrile

The polarographic behaviour of n-butylthioglycolate (RSH) at the DME in aqueous media, methanol, and acetonitrile has been investigated in the presence of 0.1 M KNO3 and 0.01% thymol. The effect of pH, concentration of RSH, and drop time on the wave characteristics and the mechanism of the reaction occurring at the surface of the mercury drop have been studied. Well-defined reversible and diffusion-controlled anodic waves were obtained in aqueous media (pH 4.2), 40% methanol (pH 3.22), and 40% acetonitrile (pH 2.96). Mathematical and analytical evidence was obtained to show that the anodic wave of RSH at a dropping mercury electrode in aqueous media, 40% methanol, and 40% acetonitrile is due to the formation of the mercury complex RSHg. The dissociation constant (pK) of the mercapto group in n-butylthioglycolate is 9.6 and the diffusion coefficient in the different media are 1.17 × 10−6 cm2 s−1 (in aqueous media) 1.23 × 10−6 cm2 s−1 (in 40% methanol), and 2.43 × 10−6 cm2 s−1 (in 40% acetonitrile). The linearity of id with RSH concentration provides a rapid and precise method for the determination of RSH, down to 0.4 mM in aqueous media, methanol, and acetonitrile.

Purification and characterization of UDP-N-acetylgalactosamine: globotriaosylceramide beta-3-N-acetylgalactosaminyltransferase, a synthase of human blood group P antigen, from canine spleen.

A UDP-N-acetylgalactosamine:globotriaosylceramide beta-3-N-acetylgalactosaminyltransferase which catalyzes the conversion of human blood group Pk antigen into P antigen has been purified over 18,000-fold in 4% yield from a Triton X-100 extract of canine spleen microsomes by affinity chromatography on UDP-hexanolamine-Sepharose and globotriaosylceramide acid-Sepharose. The purified enzyme migrates as two major bands with apparent molecular weights of 64,000 and 57,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A single band, with enzyme activity, was observed in nondenaturing acrylamide gels containing Triton X-100. Mn2+ was required for activity, and the pH optimum was 6.9. Km values for UDP-GalNAc and globotriaosylceramide were 14 and 2.5 microM, respectively. Studies on substrate specificities indicate that the preferred substrates have the general structure Gal alpha 1-4Gal-OR in which the nature of the R moiety has relatively little effect on activity. An antibody against the purified enzyme eliminated the activity of the enzyme, but did not neutralize the alpha-3-N-acetylgalactosaminyltransferase involved in the biosynthesis of Forssman glycolipid.

Selectivity and sensitivity to hydrogen ion blocking of batrachotoxin-modified sodium channels in nerve fiber membrane

Currents through batrachotoxin-modified sodium channels were measured by the voltage clamp method on the Ranvier node membrane. In experiments with replacement of Na+ in the external solution by K+ or NH 4 + the following series of permeabilities, determined as reversal potentials according to the equation of a static field, was obtained — PNa:\(P_{NH_4 }\):PK=1:0.47:0.19. The relative permeability for H+ was determined by measuring currents after replacement of Na+ in the external solution by nonpenetrating choline ions and lowering pH to 3.7–3.8. The ratio pH/pNa for sodium channels modified by batrachotoxin averaged 528±46. Modified channels were less sensitive to the blocking action of H+ than normal sodium channels. The difference in the effective values of pK of the acid group of normal and modified channels was 0.40–0.45.

Thermodynamic constants for tautomerism and ionization of pyridoxine and 3-hydroxypyridine in water–dioxane

Thermodynamic values of the ionization constants and tautomeric equilibrium constants of pyridoxine and 3-hydroxypyridine were determined in dioxane–water at 25 °C. The Born model is unable to explain the effect of the solvent on these constants. The logarithms of the equilibrium constants calculated change linearly according to the logarithm of the molar water content in the mixture, up to 40% dioxane in the case of pyridine ionizations, and up to 70% dioxane (maximum content investigated) for the phenol ionizations. The effect of protonation of either the phenolate or pyridinium nitrogen groups on the pK of the other group in these substances increases when the water content in the medium is decreased.

Importance of arginine residues in the determination of the biological activity of human corticosteroid-binding globulin

The binding activity of human corticosteroid-binding globulin (CBG) is pH dependent and governed in alkaline pH ranges by the pK of arginine. No essential arginine residues is located in the binding site. The loss of biological activity is rapid and complete as soon as one arginine residue is modified by phenylglyoxal. There is also a transconformation of the CBG molecule. Therefore the surprising stability of CBG up to pH 11.5 may be explained by a large dependence of the CBG tertiary structure on the integrity of one arginine residue : as long as the ionized state of this single residue is not changed (pH less than pK of the guanidyl group) the tertiary structure of the biologically active CBG is maintained in alkaline pH ranges.

The effect of pH on the kinetics of arylsulphatases A and B.

The effect of pH on the kinetics of rat liver arylsulphatases A and B is very similar and shows that two groups with pK values of 4.4-4.5 and 5.7-5.8 are important for enzyme activity. Substrate binding has no effect on the group with a pK of 4.4-4.5; however, the pK of the second group is shifted to 7.1-7.5 in the enzyme-substrate complex. An analysis of the effect of pH on the Ki for sulphate inhibition suggests that HSO4-is the true product. A model is proposed that involves the two ionizing groups identified in the present study in a concerted general acid-base-catalysed mechanism.

The pK of the amino terminal groups of carbonmonoxy- and deoxyhemoglobin measured by dinitrophenylation in phosphate buffers.

The rate of reaction of the terminal valines of the alpha- and beta-chains of hemoglobin with 1-fluoro-2,4-dinitrobenzene was followed spectrophotometrically at 353 nm. The variation with pH of the rate of dinitrophenylation of these groups was measured for both carbonmonoxy- and deoxyhemoglobin. In carbonmonoxyhemoglobin the results indicated a pK near 6.7 and 7.7 for the amino terminal groups of the two kinds of subunits, and were attributed to the alpha- and beta-chains respectively. Removal of ligands produced an increase of 0.1 in both pK values and a decrease of 40% of the pH-independent kinetic constant for dinitrophenylation of the beta-subunits. These modifications are due to the conformational changes associated with ligand binding in the system. In phosphate buffers the contribution to the Bohr effect of the amino terminal residues of either chains is negligible.

Ionization behavior of proteins. II. Chymotrypsinogen and its group‐modified derivatives: Study of their ionization characteristics by potentiometric and calorimetric titration

AbstractChymotrypsinogen, nitrated chymotrypsinogen (two of the four tyrosyls nitrated), acetylated chymotrypsinogen (all amino groups blocked), and nitrated‐acetylated chymotrypsinogen were titrated as f(pH) in an isoperibolic calorimeter at 20°C. After appropriate correction and reduction of both the potentiometric and thermal titration data, the parameters N (ionizable groups per group‐set), pK′, and ΔHi (heat of ionization) were evaluated using the iterative curve‐fitting algorithm of the MLAB computer program. The pK′ parameters so obtained for the two normally ionizing tyrosyl groups in chymotrypsinogen and the two nitrated tyrosyl groups in the nitrated proteins essentially agreed with the results of spectral titration. Excellent fits to all data could be obtained using evaluated parameter sets of N and pK′ for the potentiometric titration data (groups vs pH plots) and N, pK′, and ΔHi sets for the calorimetric data (total heat vs pH plots). The invocation of electrostatic interaction effects was not required to explain the data satisfactorily, despite the differences in charge number and type among the four proteins. Rather, the data can be represented by series expressions of the mass‐action law. Using all information, viz., the consequences of functional group modification, the downscale shift in tyrosyl group pK's on nitration, and the numerical values of the evaluated N, pK′, and ΔHi parameter sets for all proteins, the chemical identity of the various classes of group sets can be assigned with reasonable assurance.

The gecko visual pigment: a pH indicator with a salt effect.

1. Unlike rhodopsin, the extracted 521-pigment of the Tokay gecko (Gekko gekko) is pH-sensitive and changes its spectral absorbance in the pH range of 4.5-7.3. The colour change is reversible and pH can be employed to adjust the spectral maximum anywhere between 490 nm and its native location at 521 nm.2. The hypsochromic shift with increasing acidity is opposite to that expected for the protonation of the Schiff base nitrogen and suggests an action on the secondary system of interacting charges that have long been postulated to adjust vertebrate visual pigment colour within the visible spectrum.3. Chloride ions modulate this pH effect in a systematic and significant manner. For the pigment extracted in the chloride-deficient state the colour change occurs in the pH range of 6.0-7.0, the midpoint being close to 6.5, suggesting the possible participation of the imidazole group of histidine as the functional moiety. With added NaCl the colour shifts to the region below pH 6.2.4. The modulating action of chloride is postulated to be a conformational change of the opsin leading to a shift of the secondary interacting site from one functional group to another or else to a change in pK of a single group due to the conformational alteration of the electrostatics of the system.5. At pH values between 7.5 and 9.0 a different mechanism becomes apparent. In this region a decrease occurs in the photopigment density as well as a shift in absorbance toward the blue. This alkaline effect is readily reversed either by adding NaCl or else by lowering the pH. Along with the other protective effects of chloride these ions serve to reduce or prevent this alkaline loss in density.6. Associated with this reversible photopigment loss is a reversible appearance of a product with a maximum at about 366 nm. The spectrum of this product is like that produced by the addition of 11-cis retinal to the extract. Acidification of the alkaline preparation leads to a restitution of the photopigment as well as to a reduction of the 366-product.7. Addition of hydroxylamine to the alkaline extract in appropriate concentration inhibits the restitution of pigment-521 with acid or NaCl, but adding 11-cis retinal to the system leads to restoration of the photopigment after acidification. All the evidence suggests that product-366 is either free 11-cis retinal or else held to the opsin in a form that does not alter its spectral absorbance. The alkaline effect is therefore a disruption of the aldimine bond of the visual pigment.8. In many respects the gecko 521-pigment behaves like the chicken cone pigment, iodopsin, suggesting that an investigation of the latter in terms of pH may be a worthy project for future study.9. With its ability to change colour with pH, with chloride, with nitrate, etc. the extractable gecko pigment offers possibilities for the investigation of mechanisms responsible for adjusting visual pigment absorbance throughout the visible spectrum. The techniques of circular dichroism, Raman spectroscopy, infra-red spectroscopy, etc. may find here a suitable material for these studies.

The essential sulfhydryl group of ornithine transcarbamylases. pH dependence of the spectra of its 2-mercuri-4-nitrophenol derivative.

The essential sulfhydryl group of the ornithine transcarbamylases (ornithine carbamoyltransferase, 2.1.3.3) from bovine liver and Streptococcus faecalis reacts preferentially with 2-chloromercuri-4-nitrophenol. The spectra of this derivative between pH 4.4 AND 8.8 HAVE BEEN RESOLVED INto the spectrum of the nitrophenolate ion (III) and two species of phenol (I and II). The lambda max of I and II (both enzymes) and III (bovine) are red shifted from those of the comparable species in the same derivative of 2-mercaptoethanol. Deprotonation of a residue on the enzyme must be responsible for the transition from I to II. The pK values of the phenolic group are 7.1 (mercaptoethanol), 7.7 (bovine), and 8.8 (S. faecalis). The red shift in the lambda max of III and the modest increase in the pK of the phenolic group are consistent with a relatively hydrophobic environment for the nitrophenolate ion in the bovine enzyme. Since deprotonation of the residue in the bovine enzyme perturbs the pK of the phenolic group only slightly, its effect may be indirect. Interaction with a neighboring carboxyl group (pK 5.3) would account for the large increase in the pK of the phenolic group in the S. faecalis enzyme, which is not accompanied by an appreciable shift in the lambda max. Carbamyl-P increases the pK of the phenolic group in both enzymes, a result consistent with its binding site being close to the essential sulfhydryl group.

Kinetic analysis of a new human ornithine carbamoyltransferase variant

A new human enzymatic variant was found in a patient with ornithine carbamoyltransferase (carbamoylphosphate: l-ornithine carbamoyltransferase, EC 2.1.3.3) deficiency. This mutant enzyme has decreased affinity, with an abnormal K m value for ornithine (3–5-time greater than control at all pH values). The maximal velocity ( V) varied with pH as a normal enzyme but the sigmoid curve obtained ( V vs. pH) is shifted towards alkaline pH values. The pK of the functional catalytic group is 8.3 instead of 6.65 of a control enzyme. At its optimum pH the V of the mutant enzyme is greater than the V of the normal enzyme. Other mutant enzyme proteins with abnormal affinity for ornithine have already been described. They all are different from the reported here.

Reactivity of the Thiol Group in Human and Bovine Albumin at pH 3–9, as Measured by Exchange with 2,2′‐Dithiodipyridine

The kinetics for exchange between an aromatic disulphide and the thiol group in human and bovine albumin as well as in glutathione were investigated in the pH range 2.5--9.8. For both albumins the rate constants exhibit a maximum near pH 3, confirming the results of Svenson and Carlsson's investigation of bovine albumin [A. Svenson and J. Carlsson (1975) Biochim. Biophys Acta, 400, 433--438]. This was related to the well known N--F conformational change of the protein. At pH 5--8 the reactivity of the thiol group in both albumins and glutathione changes sharply, probably due to ionization of the thiol group. At pH above 8, however, the reactivity of the thiol group in albumins, but not in glutathione, becomes nearly independent of pH. In addition, a conformational change at pH 6.5--8.5 was studied by means of differential spectroscopy of bilirubin, liganded to human albumin. This neutral transition appeared to proceed identically in mercaptalbumin and nonmercaptalbumin. It is concluded that (a) the pK of the thiol group in albumin is significantly below that of SH in glutathione, and (b) ionization of this thiol group, Cys-34, is independent of the neutral transition.

Enzymatic synthesis of β-lactam antibiotics: A thermodynamic background

The equilibrium constants and the respective standard Gibbs energy changes for hydrolysis of some β-lactam antibiotics have been determined. Native and immobilized penicillin amidase (EC 3.5.1.11) from Escherichia coli has been used as a catalyst. The values of standard Gibbs energy changes corresponding to the pH-independent product of equilibrium concentrations ( ΔG 0 c = − RT ln K c ) have been calculated. The differences in the structure of the antibiotics nucleus hardly ever affect the value of the pH-independent component of the standard Gibbs energy change (ΔG 0 c) and value of apparent standard Gibbs energy change at a fixed pH (ΔG 0′ c). At the same time, the value of ΔG 0 c is more sensitive to the structure of the acyl moiety of the antibiotic; when ampicillin is used instead of benzylpenicillin, ΔG 0 c increases by ∼6.3 kJ mol −1 (1.5 kcal mol −1). pH-dependences of the apparent standard Gibbs energy changes for hydrolysis of β-lactam antibiotics have been calculated. The pH-dependences of ΔG 0′ c for hydrolysis of all β-lactam antibiotics have a similar pattern. The thermodynamic pH optimum of the synthesis of these compounds is in the acid pH range (pH < 5.0). The breakage of the β-lactam ring leads to a sharp decrease in the ΔG 0′ c value and a change in the pattern of the pH-dependence. For example, at pH 5.0 ΔG 0′ c decreases from 14.4 kJ mol −1 for benzylpenicillin to −1.45 kJ mol −1 for benzylpenicilloic acid. The reason for these changes is mainly a considerable increase in the pK of the amino group of the nucleus of the antibiotic and, as a consequence, a decrease in the component of standard Gibbs energy change, corresponding to the ionization of the system. The thermodynamic potentials of the enzymatic synthesis of semisynthetic penicillins and cephalosporins on the basis of both free acids and their derivatives ( N-acylated amino acids, esters) are discussed. It is shown that with esters of the acids, a high yield of the antibiotic can, in principle, be achieved at higher pH values.

Hydrogen–deuterium exchange of the C-2 protons of histidine and histidine peptides and proteins

Kinetic equations previously developed for the H–D exchange of the C-2 protons of an isolated imidazole group or for an imidazole group with an adjacent charged group of pK 6–12, have been analysed in detail. By measurement of the pseudo-first-order rate constant k for exchange in D2O as a function of pD, it is possible to obtain the secondorder rate constant k2 and the pK of the histidine residue. A nearby charged group with a pK two or more units greater than that of the histidine residue, causes a double S shaped curve when k is plotted against pD, from which the pK of the nearby charged group may be determined. The size of the second step in the curve is related to the closeness of the charged group to the histidine residue. Kinetic equations have been developed for more complex cases involving (a) two adjacent imidazole rings and one adjacent charged group of pK 6–12 and (b) an imidazole group with two adjacent charged groups of pK 6–12. The equations involve numerous second-order rate constants and equilibrium constants which cannot be evaluated explicity using experimental data obtained for L-histidyl-L-histidine, L-histidyl-L-tyrosine, and L-histidyl-L-lysine. For histidine residues in proteins it is possible to determine their pK and k2 values for H–D exchange. From the magnitude of these values one can infer the type of environment of the histidine residue. Thus the data for certain histidine residues in Iysozyme, ribonuclease, myoglobin, and leghaemoglobin have been rationalised in terms of their known environments obtained from X-ray structural studies.

The modification of the catalytic chain sulfhydryl group of aspartate transcarbamylase with mercurinitrophenols.

Aspartate transcarbamylase from Escherichia coli (EC 2.1.3.2) has four cysteines on each regulatory chain and one cysteine on each catalytic chain. The catalytic chain (C chain) thiol of aspartate transcarbamylase is resistant to most sulfhydryl reagents, but reacts rapidly and specifically with 2-chloromercuri-li-nitropheno1 (Evans, D. R., McMurray, C. H., and Lipscomb, W. N. (1972) Proc. NatZ. Acad. Sci. U. S. A. 69, 3638-3642). Here we show that the specific modification of the catalytic chain sulfhydryl residues depends on the structure of the mercurial and the pH and chloride ion concentration. A comparison of the specificity of the reaction of 14 aryl mercurials with aspartate transcarbamylase indicated that all of the reactive compounds have an ionized phenolic hydroxyl group positioned ortho to the mercury atom. The pH rate profile for 2-chloromercuri-4nitrophenol is bell shaped, exhibiting a maximum at pH 7.5. Below pH 6 and above pH 9, the mercurial reacts exclusively with the thiol groups on the regulatory chain. The rate enhancement with increasing pH on the acidic side of the optimum is probably associated with the ionization of the phenolic hydroxyl group (pK = 6.5) on the mercurial. The second order rate constant for the reaction of 2-chloromercuri-4-nitrophenol increases with chloride ion concentration to a maximum at 20 mM, but begks to decrease at chloride concentrations above 50 IXIM. The hyperreactivity of 2-chloromercuri-4-nitropheno1 is probably due to a specific binding and orientation interaction between the phenolate ion of the mercurial and either the thiol itself or a nearby positive charged residue on the enzyme. The inhibition of enzymatic activity in aspartate transcarbamylase derivatives prepared by reacting only a fraction of the catalytic chain sulfhydryl groups with nitromercurials is complex. Sites to which the mercurial is covalently bound are completely inactivated, but there is also an indirect inhibition of activity at unmodified neighboring sites. The latter type of inhibition is reversed at high concentrations of aspartate.

Calcium binding of troponin C. I. A potentiometric titration study.

Structural changes of troponin C on calcium binding were studied by hydrogen ion titration, circular dichroism, and fluorescence measurements. The potentiometric titration curves in the carboxyl region are shifted towards lower pH with calcium binding. The intrinsic pK of the carboxyl groups at the calcium binding sites decreases by 0.8 pK unit on calcium binding; on the other hand, magnesium ions have little effect on the intrinsic pK of the carboxyl groups. The intrinsic pK of the imidazole group is not affected by calcium binding. The value of w, an electrostatic interaction factor, is identical for calcium-free and calcium-bound troponin C and is about half of the value calculated assuming a compact sphere. The results of difference titration on the calcium binding indicate that the pH of troponin C solution increases on addition of CaCl2 up to 2 mol of Ca2+ per mol of troponin C and then decreases on further addition of CaCl2. The pH increase is depressed in the presence of MgCl2, in the low pH region, or at high ionic strength. The pH increase is also observed on addition of MgCl2. The ellipticity at 222 nm was measured under the same conditions as the difference titration measurements, and the relation between the pH change and the conformational change of troponin C on calcium binding is discussed based on the results obtained. The number of calcium binding sites and the binding constants estimated by analysis of these difference titration curves were in agreement with the results of Potter and Gergely (22). No magnesium binding site was observed. The tyrosine fluorescence measurements indicated that the binding site near tyrosine-109 is one of the high affinity sites.