Intercalative and nonintercalative binding of large cationic porphyrin ligands to calf thymus DNA

The large meso-substituted porphine, meso-tetra(4-N-methylpyridyl)porphine has been identified as a DNA-interactive ligand with a capacity for intercalation (1,2). Subsequently, the 2-N-methyl, 3-N-methyl and N-trimethylanilinium analogues of this porphyrin intercalator have been obtained for physico-chemical analyses (absorption spectroscopy, viscometry, circular dichroism, unwinding of supercoiled DNA). In this paper we discuss the factors affecting the character of porphyrin binding (intercalative, as is the case for the 4-N-methyl and 3-N-methyl porphines, versus non-intercalative, as is the case for the 2-N-methyl and N-trimethylanilinium porphines) and the impact that porphyrins' binding has upon the structure of DNA. The molecular conformation of the porphyrin ligand varies slightly within this series so that the ability of a given porphyrin to intercalate may be correlated with the arrangement of charged groups, the planarity of the porphine ring and the effective width of the individual molecules. The results from these studies indicate that sequence selective binding occurs within a small aperture of solution conditions.     

Intercalative and nonintercalative binding of large cationic porphyrin ligands to polynucleotides

The interactions of two positional isomers and one analogue of meso-tetra (4-N-methylpyridyl) porphine, with the synthetic polynucleotides poly[d(A-T)] . poly[d(A-T)] and poly[d(G-C)] . poly[d(G-C)] have been investigated by circular dichroism. All four porphyrins were found to bind to the polynucleotides as shown by the induction of circular dichroism in their Soret bands. Furthermore, the sign of the induced ellipticity reflects selective occupation of binding sites by the porphyrin ligands. The conformational lability of poly[d(A-T)] X poly[d(A-T)] was found to be appreciable as micromolar amounts of meso-substituted 4-N-methylpyridyl, 3-N-methylpyridyl, and p-N-trimethylanilinium porphines induced a CD spectrum similar but not identical to that of DNA in the Z-form, i.e. a negative band at 280 nm and a positive band at 259 nm. The effect of porphyrin binding to poly[d(G-C)] X poly[d(G-C)] was less pronounced and dissimilar to that seen in the AT polymer.     

CO and O2 complexes of soybean leghemoglobins: pH effects upon infrared and visible spectra. Comparisons with CO and O2 complexes of myoglobin and hemoglobin.

The effects of pH upon infrared spectra [CO stretching frequency (vco) region] and visible spectra of the CO complexes of soybean leghemoglobins a, c1, and c2, sperm whale myoglobin, and human hemoglobin A are reported. The vco for leghemoglobin--CO complexes was 1947.5 cm-1 at neutral pH. At acid pH myoglobin-- and hemoglobin--CO complexes developed vco bands at 1966--1968 cm-1, whereas leghemoglobin--CO complexes developed vco bands at approximately 1957 cm-1. All pKapp co values determined by pH-dependent variation of vco fell in the range 4.0--4.6. The pKapp co values determined from visible spectra were consistent with vco-determined values except for that of myoglobin--CO (visible pKapp co = 5.8). The pKapp co values in the 4.0--4.6 range appear to be pK values of the distal histidines, while the visible pKapp co of myoglobin--CO appears to be the pK of a group other than the distal and proximal histidines. The data are consistent with a model in which protonation of the distal histidine permits protein-free heme FeCO geometry in leghemoglobin--CO complexes but not in myoglobin-- or hemoglobin--CO complexes. Thus the heme pockets of leghemoglobins appear to be more flexible than the heme pockets of myoglobin and hemoglobin. The effects of pH upon visible spectra of the O2 complexes of soybean leghemoglobins a, c1, and c2, sperm whale myoglobin, and human hemoglobin A also are reported. pKapp o2 values of approximately 5.5 (leghemoglobins) and 4.4 (hemoglobin) are probably the pK values of the distal histidines. Comparisons of pKapp o2 values with pKapp co values indicate a more flexible heme pocket in leghemoglobins than in hemoglobin. The O2 complex of leghemoglobin c2 differed significantly from the O2 complexes of leghemoglobins a and c1 in visible spectra and titration behavior. These differences might be associated with the small structural differences in the region between the E and F helixes of leghemoglobins.     

Inhibition of mammalian glyoxalase I (lactoylglutathione lyase) by N-acylated S-blocked glutathione derivatives as a probe for the role of the N-site of glutathione in glyoxalase I mechanism

A series of twelve S-blocked and N,S-blocked glutathione derivatives has been studied as inhibitors of glyoxalase I [R)-S-lactoylglutathione methylglyoxal-lyase (isomerising), EC 4.4.1.5) from human erythrocytes. A number of new N,S-blocked glutathiones have been synthesised. Inhibition at pH 7.0, 25 degrees C was linear-competitive in all cases and the Ki values were interpreted in terms of the absence of a specific binding interaction for the N-site of the inhibitor and the absence of coupling between binding processes at N- and S-sites (the regions around the NH2 and HS groups, respectively, of GSH analogues bound to enzyme). These observations are in strong contrast to previous results with the yeast enzyme. Some Ki values were measured for yeast glyoxalase I. A special binding interaction of the phenyl groups with enzyme from both species was found for glutathione derivatives with N-acyl groups of structure -NH X CO X X X Y X Ph but not for -NH X COPh, where X and Y were variously -CH2-, -NH- and -O-. Studies were made of the range of stability of human erythrocyte glyoxalase I to pH. The pH profiles for the Ki values of S-p-bromobenzyl)glutathione and N-acetyl-S-(p-bromobenzyl)glutathione indicated no pH dependence for the latter and little, if any, for the former inhibitor. The mean Ki over the pH range 5-8.5 for S-(p-bromobenzyl)glutathione was 1.21 +/- 0.37 microM and for N-acetyl-S-(p-bromobenzyl)glutathione in the same pH range, Ki decreased from 1.45 +/- 0.26 microM to 0.88 +/- 0.11 M.     

Porphyrins and porphines bind strongly and specifically to tRNA, precursor tRNA and to M1 RNA and inhibit the ribonuclease P ribozyme reaction

Porphyrins and porphines strongly inhibit the action of the RNA subunit of the Escherichia coli ribonuclease P (M1 RNA). Meso-tetrakis(N-methyl-pyridyl)porphine followed linear competitive kinetics with pre-tRNA(Gly1) from E. coli as variable substrate (Ki 0.960 microM). Protoporphyrin IX showed linear competitive inhibition versus pre-tRNA(Gly1) from E. coli (Ki 1.90 microM). Inhibition by meso-tetrakis[4-(trimethylammonio)phenyl]porphine versus pre-tRNA(Gly1) from E. coli followed non-competitive kinetics (Ki 4.1 microM). The porphyrins bound directly to E. coli tRNAVal, E. coli pre-tRNAGly1 and M1 RNA and dissociation constants for the 1:1 complexes were determined using fluorescence spectroscopy. Dissociation constants (microM) against E. coli tRNAVal and E. coli pre-tRNAGly were: meso-tetrakis(N-methyl-pyridyl)porphine 1.21 and 0.170; meso-tetrakis[4-(trimethylammonio)phenyl]porphine, 0.107 and 0.293; protoporphyrin IX, 0.138 and 0.0819. For M1 RNA, dissociation constants were 32.8 nM for meso-tetrakis(N-methyl-pyridyl)porphine and 59.8 nM for meso-tetrakis[4-(trimethylammonio)phenyl]porphine and excitation and emission spectra indicate a binding mode with strong pi-stacking of the porphine nucleus and base pairs in a rigid low-polarity environment. Part of the inhibition of ribonuclease P is from interaction with the pre-tRNA substrate, resulting from porphyrin binding to the D-loop/T-loop region which interfaces with M1 RNA during catalysis, and part from the porphyrin binding to the M1 RNA component.     

Direct fluorometric determination of a dissociation constant as low as 10(-10) M for the subtilisin BPN'--protein proteinase inhibitor (Streptomyces subtilisin inhibitor) complex by a single photon counting technique.

It was found that an increase in fluorescence intensity at 340 nm is observed on the binding of Streptomyces subtilisin inhibitor (SSI) with subtilisin BPN' in the pH range 6--10. The dissociation constant, Ki, of the enzyme-inhibitor complex was determined as a function of pH and temperature by direct fluorometric titration utilizing the single photon counting technique in the protein concentration range of 10(-9) M. Ki values as low as 10(-10) M could be obtained with reasonable accuracy by this high-sensitivity detection method. From the temperature dependence of Ki, it was found that the binding is endothermic, and is entirely "entropy-driven" in nature. The effect of pH on Ki suggested the participation of an ionizable group with pKapp = 8.5 in the binding.     

Porphyrin with amino acid moieties: a tumor photosensitizer

A porphyrin with amino acid moieties was synthesized in this work, which may be a latent photosensitizer for photodynamic therapy (PDT). Adler's strategy was used to synthesize meso-tetra (4-nitrophenyl) porphyrin (TNPP) through cyclolization of 4-nitrobenzaldhyde and pyrrole in refluxed nitrobenzene. Reduction of TNPP yielded meso-tetra(4-aminophenyl) porphyrin (TAPP). The synthesis was improved by employing lactic acid as a catalyst. Based on TAPP, porphyrin with valine (TAPP-4Val) was obtained. The application of the resultant TAPP-4Val as tumor photosensitizer on human breast tumor cells for photodynamic therapy (PDT) was preliminarily explored. Dark-toxicity evaluations showed that, under a concentration at up to 6 x 10(-6) M, the survival of MCF-7 cells was larger than 90%, which means TAPP-4Val is almost of non-cytotoxicity. However, TAPP-4Val showed remarkable phototoxicity after visible light irradiation. Effects of irradiation time on the survival of cells under typical concentrations of TAPP-4Val were also studied. The new porphyrin with amino acid moieties, TAPP-4Val, is of high phototoxicity but minimal or no dark-toxicity, which can be used as an effective photosensitizer for PDT.     

Circular dichroism spectroscopy of a cationic porphyrin bound to DNA

Recently, the porphyrin photosensitizer meso-tetra(4-N-methyl-pyridyl)-porphine was identified as a DNA-reactive agent demonstrating both electrostatic and intercalative binding. A series of porphyrin derivatives were synthesized and studied to see if similar compounds manifested identical behavior. One derivative, meso-tetra(p-N-trimethylanilinium) porphine did not exhibit intercalation behavior but did show avid binding and novel circular dichroic features when bound to B-form DNA. At an ionic strength of 1.02, the binding constant was found to be on the order of 10⁴ and higher at lower ionic strength. The large binding constants and induced optical activity suggest that at large porphyrin/DNA ratios the final porphyrin · DNA complex may take the form of a suprahelical helix.     

Influence of glutathione on the catalytic properties of leucine aminopeptidase]

1. Leucine aminopeptidase does not catalyze the hydrolysis of glutathione. 2. Glutathione inhibits the hydrolysis of the substrates leucine hydrazide and leucine-p-nitroanilide by leucine aminopeptidase. 3. By means of kinetic experiments the type of the inhibition has been determined as noncompetitive. The inhibition constant Ki for the Mg2+-activated enzyme is five times higher than for the non-activated enzyme. 4. The degree of inhibition caused by glutathione depends on the pH value indicating a competition between glutathione and OH- ions. Mg2+-activated enzyme is invariably inhibited in the investigated pH range of 7.2 to 9.8. 5. A preincubation of the enzyme with glutathione changes the degree of activity enhancement by metal ions.     

Interaction of new pyridylporphyrins with bovine serum albumin

The interaction of meso-tetra(4-N-hydroxyethylpyridyl)porphyrin, meso-tetra(3-N-hydroxyethylpyridyl)porphyrin, and their zinc complexes with bovine serum albumin (BSA) was studied by electronic spectroscopy, CD, and equilibrium dialysis at pH 7.2. The titration of the porphyrins with BSA was accompanied by a decrease in light absorption and a bathochromic shift of the Soret band, as well as by the appearance of an isobestic point. The porphyrin interaction with BSA also led to the induction of positive CD spectra in the visible region, which is explained by the porphyrin sorption on the protein globule. The equilibrium dialysis helped in determining the stoichiometry of binding and the binding constants of the porphyrins under study with BSA using Scatchard plots. This interaction is nonspecific and reversible. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 2; see also http://www.maik.ru.     

Octa-substituted anionic porphyrins: topoisomerase I inhibition and tumor cell apoptosis induction

Beta-octabromo- meso-tetra(4-carboxyl)phenyl porphyrin 6 and beta-octaphenyl- meso-tetra(4-carboxyl)phenyl porphyrin 8 were synthesized and fully characterized by (1)H NMR, UV, and HRMS. Their cytotoxicities to tumor cells were tested using MTT assays. One kind of tumor cell apoptosis induced by these anionic porphyrins under irradiation was examined by flow cytometric analysis. The inhibition of Topo I (Topoisomerase I) indicates that Topo I preferentially binds to the synthesized compounds, thus blocking the interaction between Topo I and DNA. The results implied that compounds 4, 6, and 8 are potential inhibitors to Topo I, which might be one of the important factors inducing apoptosis of tumor cells.     

Thermodynamic and kinetic analysis of porphyrin binding to Trichosanthes cucumerina seed lectin.

The interaction of several metallo-porphyrins with the galactose-specific lectin from Trichosanthes cucumeirna (TCSL) has been investigated. Difference absorption spectroscopy revealed that significant changes occur in the Soret band region of the porphyrins upon binding to TCSL and these changes have been monitored to obtain association constants (Ka) and stoichiometry of binding (n). The dimeric lectin binds two porphyrin molecules and the presence of the specific saccharide lactose did not affect porphyrin binding significantly, indicating that the sugar and the porphyrin bind at different sites. The Ka values obtained for the binding of different porphyrins with TCSL at 25 degrees C were in the range of 2 x 10(3)-5 x 10(5) m(-1). Association constants for meso-tetra(4-sulphonatophenyl)porphyrinato copper(II) (CuTPPS), a porphyrin bearing four negative charges and meso-tetra(4-methylpyridinium)porphyrinato copper(II) (CuTMPyP), a porphyrin with four positive charges, were determined at several temperatures; from the temperature dependence of the association constants, the thermodynamic parameters change in enthalpy (DeltaH degrees ) and change in entropy (DeltaS degrees ) associated with the binding process were estimated. The thermodynamic data indicate that porphyrin binding to TCSL is driven largely by a favourable entropic contribution; the enthalpic contribution is very small, suggesting that the binding process is governed primarily by hydrophobic forces. Stopped-flow spectroscopic measurements show that binding of CuTMPyP to TCSL takes place by a single-step process and at 20 degrees C, the association and dissociation rate constants were 1.89 x 10(4) m(-1).s(-1) and 0.29 s(-1), respectively.     

Hemodextrin: a self-assembled cyclodextrin-porphyrin construct that binds dioxygen

Synthetic hemoprotein model compounds are of great interest due to the vital roles and complexities of hemoproteins. This study reports a novel, self-assembled hemoprotein model, hemodextrin. The synthesis and characterization of py-PPCD (2(A)-monopyridylmethyl-perPEGylated-beta-cyclodextrin) (2) is described. The molecular design is based on a pegylated cyclodextrin scaffold that bears both a heme-binding pocket and an axial ligand that binds an iron porphyrin. The binding constant for Fe(III)TPPS [iron(III) meso-tetra(4-sulphonatophenyl)porphyrin] by py-PPCD (2) was determined to be 2 x 10(6) M(-1) at pH 6.0 by observing characteristic changes in the UV-Vis spectrum of the porphyrin. The pyridyl nitrogen of py-PPCD (2) was shown to ligate to the iron center by observing signal changes in the Fe(II)-porphyrin 1H-NMR spectrum. This hemodextrin ensemble was shown to bind dioxygen reversibly and to form a stable ferryl species.     

Interaction of carboxypeptidase A with carbamate and carbonate esters

The carbamate ester N-(phenoxycarbonyl)-L-phenylalanine binds well to carboxypeptidase A in the manner of peptide substrates. The ester exhibits linear competitive inhibition toward carboxypeptidase A catalyzed hydrolysis of the amide hippuryl-L-phenylalanine (Ki = 1.0 X 10(-3) M at pH 7.5) and linear noncompetitive inhibition toward hydrolysis of the specific ester substrate O-hippuryl-L-beta-phenyllactate (Ki = 1.4 X 10(-3) M at pH 7.5). Linear inhibition shows that only one molecule of inhibitor is bound per active site at pH 7.5. The hydrolysis of the carbamate ester is not affected by the presence of 10(-8)-10(-9) M enzyme (the concentrations employed in inhibition experiments), but at an enzyme concentration of 3 X 10(-6) M catalysis can be detected. The value of kcat at 30 degrees C, mu = 0.5 M, and pH 7.45 is 0.25 s-1, and Km is 1.5 X 10(-3) M. The near identity of Km and Ki shows that Km is a dissociation constant. Substrate inhibition can be detected at pH less than 7 but not at pH values above 7, which suggests that a conformational change is occurring near that pH. The analogous carbonate ester O-(phenoxycarbonyl)-L-beta-phenyllactic acid is also a substrate for the enzyme. The Km is pH independent from pH 6.5 to 9 and has the value of 7.6 X 10(-5) M in that pH region. The rate constant kcat is pH independent from pH 8 to 10 at 30 degrees C (mu = 0.5 M) with a limiting value of 1.60 s-1. Modification of the carboxyl group of glutamic acid-270 to the methoxyamide strongly inhibits the hydrolysis of O-(phenoxycarbonyl)-L-beta-phenyllactic acid. Binding of beta-phenyllactate esters and phenylalanine amides must occur in different subsites, but the ratios of kcat and kcat/Km for the structural change from hippuryl to phenoxy in each series are closely similar, which suggests that the rate-determining steps are mechanistically similar.     

Excess zinc ions are a competitive inhibitor for carboxypeptidase A

The mechanism for inhibition of enzyme activity by excess zinc ions has been studied by kinetic and equilibrium dialysis methods at pH 8.2, I = 0.5 M. With carboxypeptidase A (bovine pancreas), peptide (carbobenzoxyglycyl-L-phenylalanine and hippuryl-L-phenylalanine) and ester (hippuryl-L-phenyl lactate) substrates were inhibited competitively by excess zinc ions. The Ki values for excess zinc ions with carboxypeptidase A at pH 8.2 are all similar [Ki = (5.2-2.6) X 10(-5) M]. The apparent constant for dissociation of excess zinc ions from carboxypeptidase A was also obtained by equilibrium dialysis at pH 8.2 and was 2.4 X 10(-5) M, very close to the Ki values above. With arsanilazotyrosine-248 carboxypeptidase A ([(Azo-CPD)Zn]), hippuryl-L-phenylalanine, carbobenzoxyglycyl-L-phenylalanine, and hippuryl-L-phenyl lactate were also inhibited with a competitive pattern by excess zinc ions, and the Ki values were (3.0-3.5) X 10(-5) M. The apparent constant for dissociation of excess zinc ions from arsanilazotyrosine-248 carboxypeptidase A, which was obtained from absorption changes at 510 nm, was 3.2 X 10(-5) M and is similar to the Ki values for [(Azo-CPD)Zn]. The apparent dissociation and inhibition constants, which were obtained by inhibition of enzyme activity and spectrophotometric and equilibrium dialysis methods with native carboxypeptidase A and arsanilazotyrosine-248 carboxypeptidase A, were almost the same. This agreement between the apparent dissociation and inhibition constants indicates that the zinc binding to the enzymes directly relates to the inhibition of enzyme activity by excess zinc ions. Excess zinc ions were competitive inhibitors for both peptide and ester substrates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Porphyrin accumulation in mitochondria is mediated by 2-oxoglutarate carrier

Heme (Fe-protoporphyrin IX), an endogenous porphyrin derivative, is an essential molecule in living aerobic organisms and plays a role in a variety of physiological processes such as oxygen transport, respiration, and signal transduction. For the biosynthesis of heme or the mitochondrial heme proteins, heme or its biosynthetic precursor porphyrin must be transported into mitochondria from cytosol. The mechanism of porphyrin accumulation in the mitochondrial inner membrane is unclear. In the present study, we analyzed the mechanism of mitochondrial translocation of porphyrin derivatives. We showed that palladium meso-tetra(4-carboxyphenyl)porphyrin (PdTCPP), a phosphorescent porphyrin derivative, accumulated in the mitochondria of several cell lines. Using affinity latex beads, we showed that 2-oxoglutarate carrier (OGC), the mitochondrial transporter of 2-oxoglutarate, bound to PdTCPP, and in vitro PdTCPP inhibited 2-oxoglutarate uptake into mitochondria in a competitive manner (Ki = 15 microM). Interestingly, all types of porphyrin derivatives examined in this study competitively inhibited 2-oxoglutarate uptake into mitochondria, including protoporphyrin IX, coproporphyrin III, and hemin. Furthermore, mitochondrial accumulation of porphyrins was inhibited by 2-oxoglutarate or OGC inhibitor. These results suggested that porphyrin accumulation in mitochondria is mediated by OGC and that porphyrins are able to competitively inhibit 2-oxoglutarate uptake into mitochondria. This is the first report of a putative mechanism for accumulation of porphyrins in the mitochondrial inner membrane.     

Spectrophotometric detection of pentachlorophenol (PCP) in water using immobilized and water-soluble porphyrins

The spectrophotometric properties of porphyrins are altered upon interaction with chlorophenols and other organochlorine pollutants. Meso-tetra(4-sulfonatophenyl)porphyrin (TPPS), zinc meso-tetra(4-sulfonato phenyl)porphyrin (Zn-TPPS), monosulfonate-tetraphenylporphyrin (TPPS1), meso-tri(4-sulfonatophenyl)mono(4-carboxyphenyl)porphyrin (C1TPP), meso-tetra(4-carboxyphenyl)porphyrin (C4TPP), and copper meso-tetra(4-carboxyphenyl)porphyrin (Cu-C4TPP) in solution exhibit a broad absorbance in the range 400-450 nm Soret region. The interaction of the above mentioned porphyrins in solution with pentachlorophenol (PCP) induces a red shift in the Soret spectrum with absorbance losses at 413, 418, 403, 405, 407, and 404 nm, respectively, and the appearance of new peaks at 421, 427, 431, 416, 417, and 416 nm, respectively. The intensity of the Soret spectral change is proportional to the pentachlorophenol concentration with a detection limit of 1, 0.5, 1.16, 1, 0.5, and 0.5 ppb, respectively. The interaction of (C4TPP) and (Cu-C4TPP) in solution with PCP shows to concentration dependent for concentrations less than 4 ppb the dependence was log-linear. However, for concentrations greater than 4 ppb the relation was linear. Monosulfonate-tetraphenylporphyrin immobilized as a monolayer on a Kimwipe tissue exhibits an absorbance peak in the Soret region at 422 nm. The interaction of the porphyrin with PCP induces a red shift in the Soret spectrum with absorbance loss at 419 nm and the appearance of new peaks at 446 nm. The intensity of the Soret spectral change is proportional to the log of PCP concentration. The detection limit with immobilized TPPS1 for PCP is 0.5 ppb. These results suggest the potential for development of spectrophotometric chemosensor for PCP residues in water with detection limits less than US EPA maximum contaminate level (MCL) of 1 ppb. The immobilized TPPS1 on the Kimwipe will make it possible to develop a wiping sensors to monitor the PCP or other pesticides residues on the vegetables or wood products.     

Kinetic properties of sheep brain glutathione reductase.

The kinetic properties of sheep brain glutathione reductase (GSSGR) were investigated. The enzyme showed Ping-Pong kinetics with double substrate inhibition in the forward direction. Km values for NADPH and GSSG were found to be 60.9 and 116.9 mumol/l, and Ki values were found to be 42.1 and 347.3 mumol/l, respectively. NADP+ inhibition at low fixed concentration of NADPH was mixed-type with a Ki of 281.5 mumol/l and alpha of 0.048. It is concluded that the enzyme shows a hybrid Ping-Pong-ordered branched mechanism.     

Inhibition by glutathione derivatives of bovine liver glyoxalase II (hydroxyacylglutathione hydrolase) as a probe of the N- and S-sites for substrate binding

The nature of the binding determinants used in the interaction of glutathione-based derivatives and bovine liver glyoxalase II (S-(2-hydroxyacyl)glutathione hydrolase, EC 3.1.2.6) has been investigated. Linear competitive inhibition was observed for S-blocked and S,N-blocked glutathiones with bovine liver glyoxalase II (molecular weight 22 500 by sodium dodecyl sulphate polyacrylamide gel electrophoresis; pI = 7.48 by analytical isoelectric focussing). There is a significant hydrophobic region on the enzyme to bind substituents around the sulphydryl-derived moiety of the substrate--a hydrophobic S-site. However, there is no evidence for binding of the N-site of the substrate (or inhibitor) to glyoxalase II. In contrast to glyoxalase I, there is no linkage between binding forces used at the S- and N-sites. Binding of S,N-dicarbobenzoxyglutathione is pH-dependent, showing dependence on an ionisation with pKapp approximately equal to 7.2 (binding more tightly at higher pH), as is the kcat value (pKapp approximately equal to 7.8) for S-D-lactoylglutathione.