Comparative studies on calotropins DI and DII from the latex of Calotropis gigantea

Autodigestion of two cysteine proteinases, calotropins DI and DII isolated from the latex of Calotropis gigantea, has been studied at pH 7.5 and 37 degrees C in the presence of an activating agent. Calotropin DI is more susceptible to autodigestion than calotropin DII. During autodigestion no interconversion of one calotropin to another has occurred, as verified by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. Immunologically, both calotropins are closely related, but they differ from papain and ficin. Both calotropins have blocked N-terminal amino acid residues. Their C-terminal amino acid sequences, determined by treatment with carboxypeptidase Y, are -(Pro, Ala)-Ala-Val-Tyr for calotropin DI and -(Ala, Val)-Ala-Pro-Tyr for calotropin DII. The tryptic peptide maps of their reduced and S-carboxymethylated derivatives suggest that both calotropins share a high proportion of common regions in their amino acid sequences. Calotropins DI and DII are two distinct proteinases, and they do not appear to be produced by autodigestion of a single precursor. Although they are inert to the common synthetic substrates of papain and ficin, their specificities toward oxidized insulin B chain are comparable to those of papain and ficin.     

The kinetics of the alkaline dissociation of myosin.

The rates of two processes in alkaline (pH 10.5–11.5) myosin solutions at 0 °C have been investigated: production of ionized tyrosine residues and production of light subunits. The progressive absorbance change is shown to result from a first-order irrevocable exposure to solvent and subsequent ionization of 40% of the tyrosine residues. Extrapolation to zero time gives the spectrophotometric ionization curve for native myosin; the pK of the abnormal tyrosines exceeds 12. Similarly, extrapolation to infinite time gives the curve for denatured myosin; the pK of the normal tyrosines (and of all tyrosines after denaturation) is 11.0–11.6. From the pH dependence of the rate, it is found that activation requires ionization of six residues and that their pK is much greater than 11.3. The rate of production of subunits was determined by fractionating the reaction mixture and determining the weight of light subunits produced. The process is also first order. Within experimental error, the rate constants for these two processes are equal. We conclude that they have the same rate-determining step. The data are consistent with either of two simple possible mechanisms. These are a rapid conformation change, followed by rate-determining subunit dissociation, followed by a rapid, irrevocable conformation change; or, a rapid conformation change, followed by a rate-determining, irrevocable conformation change, followed by rapid subunit dissociation.     

Characterization of lecithin:Cholesterol acyltransferase from human plasma: II. Physical properties of the enzyme

The physical properties of purified human plasma lecithin:cholesterol acyltransferase (LCAT) were investigated by techniques including analytical ultracentrifugation, ultraviolet spectroscopy, electrofocusing, and circular dichroism. The partial specific volume of LCAT was determined by sedimentation equilibrium ultracentrifugation experiments in H₂O and D₂O solutions (0.702 ml/g). The M_r was 67,000 by sodium dodecyl sulfate (SDS)-gel electrophoresis and 60,000 by sedimentation equilibrium ultracentrifugation. The discrepancy between the two sets of data presumably arose from the glycoprotein nature of the enzyme. Studies of the ultraviolet spectrum indicated that LCAT contained 6.5% ($\text{w}\text{w}$) tyrosine which corresponds to approximately 18 tyrosine residues/mol of LCAT (polypeptide M_r 45,000). Spectrophotometric titration of the ionizable phenolic side chains indicated that nearly all the tyrosine residues were buried at neutral pH while they became gradually exposed at higher pH. The apparent pK of this transition was about 12.0 contrasted with 9.8, the apparent pK of ionization of the free tyrosyl groups.     

Effect of Lipophilicity and Drug Ionization on Permeation Across Porcine Sublingual Mucosa

Sublingual route is one of the oldest alternative routes studied for the administration of drugs. However, the effect of physical-chemical properties on drug permeation via this route has not been systemically investigated. The objective of this study was to determine the effect of two key physicochemical properties, lipophilicity and ionization, on the transport of drugs across porcine sublingual mucosa. A series of β-blockers were used to study the effect of lipophilicity on drug permeation across the sublingual mucosa, while nimesulide (pKa 6.5) was used as a model drug to study the effect of degree of ionization on sublingual mucosa permeation of ionized and unionized species. Permeation of β-blockers increased linearly with an increase in the lipophilicity for the range of compounds studied. The permeability of nimesulide across sublingual mucosa decreased with an increase of pH. The flux of ionized and unionized forms of nimesulide was determined to delineate the contribution of ionized and unionized species to the total flux. At low pH, the apparent flux was primarily contributed by unionized species; however, when the pH is increased beyond its pKa, the primary contributor to the apparent flux, nimesulide, is ionized species. The contribution of each species to the apparent flux was shown to be determined by the thermodynamic activity of ionized or unionized species. This study identified the roles of lipophilicity and thermodynamic activity in drug permeation across the sublingual mucosa. The findings can help guide the design of sublingual drug delivery systems with optimal pH and solubility.     

Crystal and molecular structure of the sulfhydryl protease calotropin DI at 3·2 Å resolution

The three-dimensional structure of the sulfhydryl protease calotropin DI from the madar plant, Calotropis gigantea, has been determined at 3·2 Å resolution using the multiple isomorphous replacement method with five heavy atom derivatives. A Fourier synthesis based on protein phases with a mean figure of merit of 0·857 was used for model building. The polypeptide backbone of calotropin DI is folded to form two distinct lobes, one of which is comprised mainly of α-helices, while the other is characterized by a system of all antiparallel pleated sheets. The overall molecular architecture closely resembles those found in the sulfhydryl proteases papain and actinidin.Despite the unknown amino acid sequence of calotropin DI a number of residues around its active center could be identified. These amino acid side-chains were found in a similar arrangement as the corresponding ones in papain and actinidin. The polypeptide chain between residues 1 and 18 of calotropin DI folds in a unique manner, providing a possible explanation for the unusual inability of calotropin DI to hydrolyze those synthetic substrates that papain and actinidin act upon.     

Spectrophotometric titration of phenolic groups of pepsin.

The ionization of tyrosine residues in diazotized pepsin under various solvent conditions was studied. All tyrosyl residues of the protein titrated normally with a pK of 10.02 in 6 M guanidine hydrochloride solution. On the other hand, two stages in the phenolic group titration curve were observed for the inactivated protein in the absence of guanidine hydrochloride; only about 10 tyrosine residues ionized reversibly up to pH 11, above which titration was irreversible. The irreversible titration zone corresponds to the pH range 11--13 in which unfolding, leading to the random coil state, was shown to occur by circular dichroism and viscosity measurements. The number of tyrosine residues exposed in the native and alkali-denatured (pH 7.5) states of diazotized protein were also studied by solvent perturbation techniques; 10 and 12 groups are exposed in the native and denatured states, respectively.     

Alkali-induced reduction of the b-cytochromes in purified Complex III from beef heart mitochondria

Approx. 40–50% of the cytochrome b in purified Complex III is reduced by ascorbate plus N,N,N′,N′-tetramethyl-p-phenylenediamine or phenazine methosulfate at neutral pH. The remaining cytochrome b, including cytochrome b-565, is reduced by increasing the pH. The apparent pK for this reduction is between pH 10 and 11, and is more than two pH units higher than a similar alkali-induced transition in Mg-ATP particles. Alkali-induced reduction of cytochrome b occurs concomitantly with the exposure of hydrophobic tyrosine and tryptophan residues to a more hydrophilic environment. The relationship of these findings to the presence of a substrate accessibility barrier in Complex III is discussed.     

Analysis of the ionization constants and heats of ionization of reduced and oxidized horse heart cytochrome c

Simultaneous curve fitting for the ionization parameters of oxidized and reduced horse heart cytochrome c in 0.15M KCl and 20°C yields values for the ionization constants (as pK′) and the heats of ionization (ΔH_i) which can reconstruct either the potentiometric or thermal titration curves. Reduced cytochrome c requires 8 sets of groups, whereas oxidized cytochrome c requires 10 sets of groups. The additional groups in the oxidized preparation appear to involve the ferriheme (pK′, 9.25; ΔH_i, 13.7 kcal/mol) and a tyrosine (pK′ ? 10.24) that is not present in the reduced form. The potentiometric and thermal difference curves (reduced – oxidized) involve the appearance of 17 kcal/mol centered at pH 9.7 and 5.8 kcal/mol centered at pH 4.9. The carboxyl groups in both species appear to be normal for the hydrogen-bonded form. Only one histidine has normal ionization properties (pK′, 6.7; ΔH_i, 7.5 kcal/mol), as do 17 of the lysine residues (pK′, 10.8; ΔH_i, 11.5 kcal/mol).     

pH dependence of conformational fluctuations of the protein backbone

Proton binding equilibria (pK_a values) of ionizable groups in proteins are exquisitely sensitive to their microenvironments. Apparent pK_a values measured for individual ionizable residues with NMR spectroscopy are actually population‐weighted averages of the pK_a in different conformational microstates. NMR spectroscopy experiments with staphylococcal nuclease were used to test the hypothesis that pK_a values of surface Glu and Asp residues are affected by pH‐sensitive fluctuations of the backbone between folded and locally unfolded conformations. ¹⁵N spin relaxation studies showed that as the pH decreases from the neutral into the acidic range the amplitudes of backbone fluctuations in the ps‐ns timescale increase near carboxylic residues. Hydrogen exchange experiments suggested that backbone conformational fluctuations promoted by decreasing pH also reflect slower local or sub‐global unfolding near carboxylic groups. This study has implications for structure‐based pK_a calculations: (1) The timescale of the backbone's response to ionization events in proteins can range from ps to ms, and even longer; (2) pH‐sensitive fluctuations of the backbone can be localized to both the segment the ionizable residue is attached to or the one that occludes the ionizable group; (3) Structural perturbations are not necessarily propagated through Coulomb interactions; instead, local fluctuations appear to be coupled through the co‐operativity inherent to elements of secondary structure and to networks of hydrogen bonds. These results are consistent with the idea that local conformational fluctuations and stabilities are important determinants of apparent pK_a values of ionizable residues in proteins. Proteins 2014; 82:3132–3143. © 2014 Wiley Periodicals, Inc.     

Carboxyl pKa Values and Acid Denaturation of BBL

The protein BBL undergoes structural transitions and acid denaturation between pH 1.2 and 8.0. Using NMR spectroscopy, we measured the pK_a values of all the carboxylic residues in this pH range. We employed ¹³C direct-detection two-dimensional IPAP (in-phase antiphase) CACO NMR spectroscopy to monitor the ionization state of different carboxylic groups and demonstrated its advantages over other NMR techniques in measuring pK_a values of carboxylic residues. The two residues Glu161 and Asp162 had significantly lowered pK_a values, showing that these residues are involved in a network of stabilizing electrostatic interactions, as is His166. The other carboxylates had unperturbed values. The pH dependence of the free energy of denaturation was described quantitatively by the ionizations of those three residues of perturbed pK_a, and, using thermodynamic cycles, we could calculate their pK_as in the native and denatured states as well as the equilibrium constants for denaturation of the different protonation states. We also measured ¹³C^α chemical shifts of individual residues as a function of pH. These shifts sense structural transitions rather than ionizations, and they titrated with pH consistent with the change in equilibrium constant for denaturation. Kinetic measurements of the folding of BBL E161Q indicated that, at pH 7, the stabilizing interactions with Glu161 are formed mainly in the transition state. We also found that local interactions still exist in the acid-denatured state of BBL, which attenuate somewhat the flexibility of the acid-denatured state.     

Induction of high phenolic pK of iodothyronines by sodium dodecyl sulfate: Adaptation to quantitative analysis of the iodoamino acid content of thyroglobulin

Sodium dodecyl sulfate (SDS) increased the pK value of thyroxine from 6.7 to 8.5 and that of 3,3′,5-triiodothyronine from 8.3 to 9.8. The pK, however, of monoiodotyrosine and diiodotyrosine was not affected. This shift in ionization with SDS, when measured at pH 8.0, shows a charaeteristic negative spectral difference with maximum values at 332 and 326 nm for thyroxine and triiodothyronine, respectively. The difference in absorption is pH-dependent with maxima at pH 8.0 and 9.5 for thyroxine and triiodothyronine, respectively. The increase in pK value was dependent on the SDS concentration between 0.01% (0.35 mM) and 0.05% (1.8 mM) and little further change was observed above 0.1% (3.5 mM).A similar negative difference in absorption was observed at approx. 320 nm when SDS was added to thyroglobulin solutions at pH 8.0. No negative difference was observed in this wavelength region with thyroglobulin lacking iodoamino acid residues. When the SDS concentration was above 0.1% (3.5 mM), the value of the negative difference depended on the number of iodoamino acid residues present in thyroglobulin. The magnitude of the negative band of thyroglobulin was dependent on the SDS concentration between 0.01% and 0.1%, and was invariant above 0.1% SDS.     

The pH jump study of enzyme proteins. I. Liquefying alpha-amylase from Bacillus subtilis.

Rapid conformational changes due to pH jump were studied kinetically at 25 degrees mainly by the stopped-flow method using liquefying alpha-amylase from Bacillus subtilis [EC 3.2.1-.1, liquefying]. First, the conformational change due to a pH jump produced by mixing with alkali was monitored as a function of time at 245 nm through the ionization of phenolic hydroxyl groups of tyrosine residues which were originally buried and finally become exposed due to the pH jump. Three distinct phases of conformational change were clearly recognized by this method by varying the final pH values. Each phase involved the exposure of an essentially definite number of tyrosine residues, whose rate constant was crucially dependent on pH. Second, these phases of conformational change were subjected to examination in terms of the optical rotation change at 411 nm and the reversibility upon reverse pH jump with respect to conformational reconstitution, as observed through the protonation ofphenolic hydroxyl groups of ionized tyrosine residues and the enzyme activity. The first phase, which occurs above pH 12.5, involves no change in the optical rotation and is reversible as observed by the above two monitoring methods. In contrast, the other two phases, which are observed above pH 12.7, are accompanied by an optical rotation change and no appreciable reversibility was detected by these methods.     

Human placental alkaline phosphatase: Effects on conformation by ligands which alter catalytic activity

The conformation of human placental alkaline phosphatase (EC 3.1.3.1) has been studied using the spectroscopic structural probes of pH difference spectroscopy, solvent perturbation difference spectroscopy, and circular dichroism. Of the 37 ± 1 tyrosine residues in placental alkaline phosphatase (PAP), 5 ± 1 residues are observed by pH difference spectroscopy to be “free” and presumed to be located on the surface of the enzyme molecule. The ionization of these 5 “free” tyrosyl groups is not time dependent and is reversible with a pK_app of 10.29. The remaining 32 ± 1 tyrosines are considered “buried” and ionization is observed to be both time dependent and irreversible. Treatment of the enzyme with 4 m guanidine-hydrochloride normalizes all 37 ± 1 tyrosine residues (pK_app = 10.08). The difference pH titration studies thus provide spectrophotometric evidence for a change in molecular conformation of PAP in the pH region of 10.5. Using solvent perturbation difference spectroscopy and circular dichroism, the local environments of tyrosine and tryptophan residues were elucidated for the native enzyme and the enzyme in the presence of ligands that influence catalytic function: inorganic phosphate (competitive inhibitor), l-phenylalanine (uncompetitive inhibitor), d-phenylalanine (noninhibitor). and Mg²⁺ ion (activator). The spectral observations from these studies led to the following interpretations: (i) the binding of inorganic phosphate, a competitive inhibitor, induces a conformational change in the enzyme that may alter the active site and thereby decrease enzyme catalytic function; (ii) perturbation with l-phenylalanine gives spectral results indicating a conformational change consistent with the postulate that this uncompetitive inhibitor prevents the dissociation of the phosphoryl enzyme intermediate; and (iii) Mg²⁺ ion causes a slight separation of the enzyme subunits, which could increase accessibility to the active site and, thus, enzyme activity.     

Comprehensive determination of protein tyrosine pKa values for photoactive yellow protein using indirect 13C NMR spectroscopy

Upon blue-light irradiation, the bacterium Halorhodospira halophila is able to modulate the activity of its flagellar motor and thereby evade potentially harmful UV radiation. The 14 kDa soluble cytosolic photoactive yellow protein (PYP) is believed to be the primary mediator of this photophobic response, and yields a UV/Vis absorption spectrum that closely matches the bacterium's motility spectrum. In the electronic ground state, the para-coumaric acid (pCA) chromophore of PYP is negatively charged and forms two short hydrogen bonds to the side chains of Glu-46 and Tyr-42. The resulting acid triad is central to the marked pH dependence of the optical-absorption relaxation kinetics of PYP. Here, we describe an NMR approach to sequence-specifically follow all tyrosine side-chain protonation states in PYP from pH 3.41 to 11.24. The indirect observation of the nonprotonated ¹³C_γ resonances in sensitive and well-resolved two-dimensional ¹³C-¹H spectra proved to be pivotal in this effort, as observation of other ring-system resonances was hampered by spectral congestion and line-broadening due to ring flips. We observe three classes of tyrosine residues in PYP that exhibit very different pK_a values depending on whether the phenolic side chain is solvent-exposed, buried, or hydrogen-bonded. In particular, our data show that Tyr-42 remains fully protonated in the pH range of 3.41–11.24, and that pH-induced changes observed in the photocycle kinetics of PYP cannot be caused by changes in the charge state of Tyr-42. It is therefore very unlikely that the pCA chromophore undergoes changes in its electrostatic interactions in the electronic ground state.     

O-carboxamidomethyl tyrosine as a reaction product of the alkylation of proteins with iodoacetamide

Iodoacetamide-1-¹⁴C was found to be rapidly incorporated into RBP, a protein devoid of free SH, under conditions similar to those normally employed for the derivatization of SH. The reaction extent is pH dependent and at pH 10.0 one mole of acetamide was incorporated per mole of tyrosine residue. The amino acid composition of the alkylated RBP was found to be identical to RBP except for a loss of tyrosine and the appearance of a new dicarboxylic acid peak which was converted to tyrosine by prolonged acid hydrolysisUnder similar reaction conditions, phenol and p-cresol reacted rapidly to form phenoxyacetamide and p-cresoxyacetamide. These phenolic ethers as well as anisole and phenetole were found to be readily hydrolized under the conditions normally used to hydrolyze protein.The incorporation of acetamide into RBP did not effect its riboflavinbinding capacity or its immunological reactivity to RBP antibody. The ¹⁴C alkylated RBP has been found to be a convenient tool for biological half-life studies.The tyrosine residues of glucagon react with iodoacetamide in a similar fashion and the use of ¹⁴C-iodoacetamide may prove to be a convenient means of introducing ¹⁴C into proteins. Iodoacetamide in the pH 7–10.0 range will derivatize the cysteine and tyrosine groups of proteins at comparable rates.     

The effect of pH and temperature on the kinetics of native and altered glycogen phosphorylase.

Data on the effect of pH and temperature on the kinetics of rabbit muscle phosphorylases a and b and reduced phosphorylase b (α-1,4-glucan:orthophosphate glucosyltransferase, EC 2.4.1.1) with glycogen as the saturating and inorganic phosphate the variable substrate are presented. The kinetic profiles as a function of pH are similar for these enzyme species except that the positions of the pH-maximal velocity profiles for reduced phosphorylase b are relatively invariant in the 15 °–30 ° range, whereas the “native” phosphorylases exhibit a substantial shift of the lower pH limb of the profile toward the acid side when the temperature is lowered from 30 to 15 °C. It is proposed that a group with a pK near 6.0 at 30 °C determines the acid limb of maximal velocity profiles. The phosphoryl moiety of enzyme bound pyridoxal 5′-phosphate is suggested for this group. A conformational transition in the protein, which is somehow modified when the aldimine bond between protein and pyridoxal 5′-phosphate is reduced, is invoked to account for the large decrease of this acid side apparent pK for the ternary complex of native phosphorylases when the temperature is lowered. A group with a pK near 7.1 and a heat of ionization of about 8000 cal/mol determines the alkaline limb of maximal velocity profiles at 30 °C. An imidazoyl ring ionization of an enzyme histidyl group is proposed to account for this behavior. In the enzyme-glycogen binary complex, the apparent heat of ionization of this group has an anomalous value of about ?10,000 cal/ mol. It is suggested that a neighboring amino or arginyl guanidinium group is able to interact with the imidazoyl ring in the absence of bound inorganic phosphate to cause this anomalous behavior. The effect of pH on K_m for inorganic phosphate is simply explained by a group with a pK of 6.56 and low heat of ionization. The data are interpreted to indicate that the dianion of inorganic phosphate is the true substrate for all forms of phosphorylase. The kinetic results of this report are closely compared with other kinetic data in the literature on mammalian, plant, and bacterial α-glucan phosphorylases and general overall similarity is demonstrated. Various methods for analyzing pH-kinetic data for enzymes are briefly discussed, and the crucial difference in conclusions the choice of method can make is demonstrated with our data.     

Crystallizations and preliminary X-ray studies of calotropins DI and DII

Crystals of calotropin DI (M_r 23,400), have been prepared by microdialysis against 5% (w/v) polyethylene glycol 20,000 in water, pH 7.0. They have orthorhombic space group P2₁2₁2₁ with cell parameters $\text{a = 57.5}\text{A}\text{?}\text{, b = 86.2}\text{A}\text{?}\text{, c = 40.3}\text{A}\text{?}$. Crystals of calotropin DII (M_r 24,000), prepared by the same technique against 5% (w/v) polyethylene glycol 20,000 in phosphate buffer of low ionic strength, pH 7.0, display monoclinic space group C2 with cell parameters $\text{a = 135.8}\text{A}\text{?}\text{, b = 32.0}\text{A}\text{?}\text{, c = 47.7}\text{A}\text{?}\text{, β = 103.80 °}$. In both cases, there is only one molecule in the asymmetric unit.     

Reactions of the amino groups of RNAse A: III. The existence of pH-dependent values of pK

The rates of the trinitrophenylation of the amino groups of ribonuclease A (RNAse) with the specific reagent trinitrobenzene sulfonic acid have been studied at 27°C, between pH 7.0 and 9.9. From the variation of the velocity constants with pH it has been shown that the reaction is biphasic in the sense that for each amino group two pKs have been found: one (pK = 7.3–7.52) in the range of pH between 7.0 and 8.3 and the other (pK = 9.28–9.69) in the pH range 8.5–9.9. It is pointed out that when the experimental conditions approached one another, there was agreement between the pK values obtained from titrimetric and kinetic studies. Evidence is presented from the literature concerning the validity of the pK value near 7.5 for the ε-amino groups in RNAse. The studies were repeated with performic acid oxidized RNAse and the 10 ε-amino groups were found to be monophasic with pK values between 8.01 and 8.10. The α-amino group of the N-terminal lysine was biphasic with a pK of 7.26 (pH range 7–8) and 8.13 (pH range 8.2–9.5).     

Antibody recognition and neutralization determinants on domains I and II of West Nile Virus envelope protein

Previous studies have demonstrated that monoclonal antibodies (MAbs) against an epitope on the lateral surface of domain III (DIII) of the West Nile virus (WNV) envelope (E) strongly protect against infection in animals. Herein, we observed significantly less efficient neutralization by 89 MAbs that recognized domain I (DI) or II (DII) of WNV E protein. Moreover, in cells expressing Fc gamma receptors, many of the DI- and DII-specific MAbs enhanced infection over a broad range of concentrations. Using yeast surface display of E protein variants, we identified 25 E protein residues to be critical for recognition by DI- or DII-specific neutralizing MAbs. These residues cluster into six novel and one previously characterized epitope located on the lateral ridge of DI, the linker region between DI and DIII, the hinge interface between DI and DII, and the lateral ridge, central interface, dimer interface, and fusion loop of DII. Approximately 45% of DI-DII-specific MAbs showed reduced binding with mutations in the highly conserved fusion loop in DII: 85% of these (34 of 40) cross-reacted with the distantly related dengue virus (DENV). In contrast, MAbs that bound the other neutralizing epitopes in DI and DII showed no apparent cross-reactivity with DENV E protein. Surprisingly, several of the neutralizing epitopes were located in solvent-inaccessible positions in the context of the available pseudoatomic model of WNV. Nonetheless, DI and DII MAbs protect against WNV infection in mice, albeit with lower efficiency than DIII-specific neutralizing MAbs.     

Interaction of carboxypeptidases A and B with nitrotyrosyl and aminotyrosyl derivatives of the carboxypeptidase inhibitor from potatoes

The single tyrosine residue of the carboxypeptidase inhibitor from potatoes, which is in contact with carboxypeptidase A in the enzyme-inhibitor complex as determined by X-ray diffraction. was converted to 3-nitrotyrosine by treatment with tetranitromethane in buffers containing 75% ethanol. The nitroinhibitor bound both bovine carboxypeptidase A and porcine carboxypeptidase B with apparent K_i values indistinguishable from those of the unmodified inhibitor. Spectral titration indicated that the nitrotyrosyl residue of the inhibitor ionized with pK_a of 7.25 either in the presence or absence of carboxypeptidase A; however, this pK_a was shifted to (ca 7.7 in the presence of carboxypeptidase B. Reduction of the 3-nitrotyrosine residue to 3-aminotyrosine slightly increased the strength of binding to both carboxypeptidases. These data suggest that the tyrosine residue of the inhibitor, is in a polar environment in the enzyme-inhibitor complex and that it is not involved in hydrogen bonding.