Determination of dissociation constants of crystalline-alpha-chymotrypsin complexes

As a first step in investigations of the properties of crystalline enzymes, the binding of indole, N-formyl-l-phenylalanine, and N-formyl-l-p-iodophenylalanine to α-chymotrypsin crystals, and the binding of indole to tosyl-α-chymotrypsin crystals, has been studied. The methods used were spectrophotometric measurements of the concentration of indole in the supernatant, or measurements of the concentration of radioactively labeled indole in both the supernatant and the crystal. The dissociation constants of the specific binding site of the crystalline enzyme have been determined for indole and N-formyl-l-phenylalanine. It was found that indole does not bind to tosyl-α-chymotrypsin crystals and that N-formyl-p-iodophenylalanine does not bind to the substrate binding site of the crystalline enzyme.The information obtained from these simple equilibrium measurements is in agreement with X-ray diffraction studies. The approach is, therefore, capable of determining whether or not compounds bind to the active site of a crystalline enzyme, and whether the occupancy of this site is sufficient for structure determinations using X-ray diffraction methods.     

Determination of the stability constants for the binding of sulfonated morin with Fe

The complexation of the sodium salt of sulfonated morin (H₅SM⁻) with Fe²⁺ was studied by potentiometric titration as was its deprotonation. Only four of the five hydroxy groups were deprotonated under the conditions employed. The associated pK_a values are 3.80, 7.47, 9.24 and 11.48. Analysis of the titration data suggests formation of (H₃SM)Fe⁻, (H₂SM)Fe²⁻, (HSM)Fe³⁻ and (HSM)₂Fe⁸⁻. Log β values (based on HSM⁵⁻ as the ligand species) are 24.8, 16.1, 7.1 and 11.6, respectively. Theoretical calculations predict that the 7-hydroxy group is deprotonated first followed closely by the 3-hydroxy position. Deprotonation of the 2′-hydroxy group results in proton migration from the 3-hydroxy oxygen atom. These calculations along with previous results suggest that chelation of the metal ion likely occurs at the 3-hydroxy-4-keto site.     

The stability constants of some metal ion complexes of 3',5'-cyclic AMP.

The stability constants of complexes of 3', 5'-cyclic AMP with Mg2+, Ca2+, Mn2+, Ni2+ and Co2+ were estimated at 30 degrees C in solutions of ionic strength about 0.15 containing about 130 mM K+ or tetramethylammonium ions. Values between 13 and 22 M-1 were obtained, indicating that only about 2% of cyclic AMP is present as metal complexes in vivo, but that at commonly used in vitro concentrations of 10 mM bivalent metal ions, 10--20% of cyclic AMP is present as metal complexes. The possible significance of these metal complexes, for example as competitive inhibitors, is discussed.     

Determination of the stability constants and oxidation susceptibility of nickel(II) complexes with 2'-deoxyguanosine 5'-triphosphate and L-histidine

The formation of binary Ni(II) complexes with 2'-deoxyguanosine 5'-triphosphate (dGTP, L) as well as ternary complexes thereof with L-histidine (His, A) was studied with the use of potentiometry and electronic absorption spectroscopy. In the binary and ternary systems, the complexes with stoichiometries NiH2L-, NiHL2-, NiL3- and NiH2LA2-, NiHLA3-, NiLA4- respectively, were detected. The ternary complexes are very stable at pH 7.4 and thus may constitute biologically relevant Ni(II) carriers in the cell. In the presence of hydrogen peroxide, the binary and ternary systems both generate hydroxyl radical-like species and undergo dGTP degradation with the formation of the 8-oxo-dGTP intermediate. The latter, along with dGTP complexation and degradation, may lead to mutagenesis and carcinogenesis due to base-mispairing properties of 8-oxoguanine and the disturbance in the physiological balance among the four canonical triphosphodeoxynucleotide substrates for DNA synthesis.     

Apparent stability constants of H+ and Mg2" complexes of 5-phosphoribosyl alpha-1-pyrophosphate.

Apparent Mg2+ and H+ stability constants of 5-phosphoribosyl alpha-1-pyrophosphate (ligand, L) complexes were determined from pH titration data at 25 degrees C with an average of 0.17 M NaCl or KCl and 0.20 M ionic strength. The logarithms of calculated macroscopic overall stability constants are: 3.2 (MgL3-), 4.8 (Mg2L-), 6.5 (HL4-), 12.4 H2L3-), 9.4 (Mg HL2-), and 11.0 (MgH2L). Comparison of the stepwise Mg2+ stability constants (log k = 3.2 and 1.6) with those of MgADP- and MgAMP or Mg-hexose-1-P suggests that the first and second Mg2+ bind to the 1-PP and 5-P groups of the ligand, respectively. Reasonable assumptions about relative microscopic constants indicate that several of the microscopic isomers do not achieve significant concentrations over a large range of conditions. Judging from other data on organophosphate complexes, it is likely that the constants of this study may be extrapolated with little error to other conditions of ionic strength 0.1--0.2 M) and temperature (e.g., 15--35 degrees C), and widely different monovalent ion concentrations.     

Determination of individual cell Michaelis-Menten constants.

BACKGROUND: A novel methodology for the measurement and analysis of apparent K(M) (Michaelis-Menten constant) and V(MAX) values of individual cells is suggested. It is based on a mathematical model that considers substrate influx into the cell, its intracellular enzymatic hydrolysis, and the product efflux. The mathematical formulation was approximated linearly in order to analyze intracellular substrate conversion characteristics via Michaelis-Menten theory. METHODS: Utilizing static cytometry, the time dependence of the fluorescence intensity [FI(t)] emitted from prelocalized and defined FDA stained cells was recorded. This required frequent periodical measurements of the same cells, which are sequentially exposed to various fluorogenic substrate concentrations. RESULTS: Model simulations correlated with experimental results. Differences in distributions of individual K(M) and V(MAX) values of cells incubated with and without PHA were evident. Average K(M) and V(MAX) values of PHA-stimulated cells increased by 99% and 540%, respectively. CONCLUSIONS: This study may provide a tool for assessing intracellular enzymatic activity in individual intact cells under defined physiologic conditions. This may open new vistas in various areas, giving answers to critical questions arising in the field of cell and developmental biology, immunology, oncology, and pharmacology.     

Potentiometric measurement of stability constants of complexes between fulvic acid carboxylate and Fe3+

The stability constants of complexes formed between iron (III) and fulvic acid extracted from organic manures and wastes such as urban domestic sewage sludge, farmyard manure, poultry manure and sulfitation pressmud were investigated by the potentiometric titration method in an ionic medium of 0.1 M KNO₃ at 25±1 °C. A modification of the Katchalsky's model was employed for the estimation of stability constants. The displacement of the titration curves due to presence of Fe³⁺ in FA solutions formed the basis of calculations. The weak acidic property of fulvic acids due to carboxyl groups resulted in buffering over a wide range of pH; fulvic acids were completely neutralized in the pH range of 7.00–8.85. Apparent dissociation constants (pK_APP) of weakly acidic carboxyl groups were a direct function of degree of dissociation (α_L) in the mid-range of titration curves but were non-linear at high and low α_L values. The stability constants for formation of Fe–FA complexes (log β_Fe) calculated from the titration data were in the range of 5.64–7.55, depending upon α_L and electrostatic properties of fulvic acids. The relatively high stability constants of Fe–FA complexes in comparison to those with other competing cations suggest that the Fe–FA complexes are relatively stable in a soil environment. This revised version was published online in June 2006 with corrections to the Cover Date.     

Carbamoylphosphonate-based matrix metalloproteinase inhibitor metal complexes: solution studies and stability constants. Towards a zinc-selective binding group

Overactive matrix metalloproteinases (MMPs) are associated with a variety of disease states. Therefore, their inhibition is a highly desirable goal. Yet, more than a decade of worldwide activity has not produced even one clinically useful inhibitor. Because of the crucial role of zinc in the activity of the enzyme, the design of inhibitors is usually based upon a so-called zinc binding group (ZBG). Yet, many of the hitherto synthesized potent inhibitors failed clinically, presumably because they bind stronger to metals other than zinc. We have developed in vivo potent inhibitors based on the carbamoylphosphonic group as a putative ZBG. In this paper we report stability constants for Ca(II), Mg(II), Zn(II) and Cu(II) complexes of two potent, in vivo active, MMP inhibitors, cyclopentylcarbamoylphosphonic acid (1) and 2-(N,N-dimethylamino)ethylcarbamoylphosphonic acid (2). Precipitation prevented the determination of stability constants for iron(III) complexes of 1 and 2. For comparison with carbamoylphosphonates 1 and 2, we synthesized 2-cyclohexyl-1,1-difluoroethylphosphonic acid (3), which does not inhibit MMP, and determined the stability constants of its complexes with Mg(II), Ca(II) and Zn(II). Comparison with the values obtained from the complexes of 1 and 2 with those from 3 indicates participation of the C=O group in the metal binding of the former compounds. The complex stability orders for both 1 and 2 are Ca(II)<Mg(II)<Zn(II)<Cu(II). In addition, the results indicate that at pH>8 the dimethylamino group of compound 2 can also participate in the binding of the transition metals Cu and Zn. On the other hand, the amino group in carbamoylphosphonic acid 2 lowers the stability of the complexes with metals favoring oxygen ligands (Ca, Mg and Fe) and increases the selectivity towards Zn. These results are helpful for rationalizing the results observed on our MMP inhibitors hitherto examined, and are expected to be useful for the design of new selective inhibitors.Electronic Supplementary Material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00775-004-0524-5     

Improved methods for calculating formation constants for nucleotide-cation complexes

Two data reduction methods that can be used to calculate formation constants for nucleotidecation complexes are described. Both methods are used to analyze data obtained by an anion-exchange resin method, and either method can improve the accuracy of the calculated formation constants by more than 50%. The key to this significant improvement in accuracy is the realization that the equation for the mathematical model describing such systems is always nonlinear in terms of the formation constants and, in the general case, is higher order in the cation concentration. There are two major reasons for the improved quality of the results associated with the new model. First, successive linear extrapolations are eliminated, and error propagation is reduced. Second, all of the data are used for the simultaneous calculation of all formation constants, and the uncertainty due to random experimental errors is minimized.     

Determination of the viscometric constants for chitosan

The viscometric constants a and K_m in the Mark-Houwink equation have been determined for chitosan in 0.1 m acetic acid 0.2 m sodium chloride solution, using the approach of Sharples and Major. The number-average molecular weights were determined by absorbance measurements on solutions of the phenylosazone derivatives. The values obtained a = 0.93, K_m = 1.81 × 10^?3 cm³ g¹ differ considerably from those reported previously by Lee but are in agreement with values found for other ionic polysaccharides having related β-(1 → 4)-linked structures.     

The stability constants of copper(II) complexes with some alpha-amino acids in dioxan-water mixtures

In this study, the overall stability constants of copper(II) complexes with some alpha-amino acids (glycine, dl-alanine, dl-valine, l-leucine, l-asparagine, l-glutamine) were determined by potentiometric titration in water, 25% dioxan-75% water, 35% dioxan-65% water, 50% dioxan-50% water, and 60% dioxan-40% water. The titrations were performed at 25 degrees C, under nitrogen atmosphere, and the ionic strength of the medium was maintained at 0.10 M by using sodium perchlorate. The formation curves of their complexes (n-p[L]) were obtained by means of the titration data. Then the stability constants were determined in relation to these curves. The mol ratio of copper(II) to alpha-amino acid was also determined and it was found that the complexes were CuL(2) type. Another important result obtained was that the tendency of amino acids to form complexes with copper(II) was greater in dioxan-water mixtures compared to water.     

Determination of the mechanism and kinetic constants for hog kidney gamma-glutamyltransferase.

The initial-velocity kinetics of hog kidney gamma-glutamyltransferase were studied. Glutamate gamma-(4-nitroanilide) and its 3-carboxy derivative, glutamate gamma-(3-carboxy-4-nitroanilide), served as gamma-glutamyl donors, and glycylglycine as an acceptor. Reaction products were identified by paper chromatography and amino acid analysis. Inhibited Ping Pong mechanisms and a comprehensive initial- velocity expression were developed which account for the observed simultaneous gamma-glutamyl transfer and autotransfer, competitive inhibition by glycylglycine, and non-competitive inhibition by the carboxy donor. The validity of the proposed Ping Pong mechanisms are supported by enzyme-velocity data obtained with constant ratios of acceptor to donor concentrations. Kinetic constants were determined by a non-linear regression analysis. With glutamate gamma-(4-nitroanilide) as the donor, Michaelis constants for the donor, acceptor and donor-acting-as-acceptor are 1.87, 24.9, and 2.08 mM respectively. With glutamate gamma-(3-carboxy-4-nitroanilide) as the donor, these Michaelis constants are 1.63, 16.6, and 12.3 mM. Glyclyglycine competitive inhibition constants with the parent donor and its carboxy derivative are 275 and 205 mM respectively; the non-competitive inhibition constant of the carboxy donor is 34 mM.     

Determination of association and dissociation rate constants for bilirubin--bovine serum albumin.

The association rate constant for the binding of bilirubin to bovine serum albumin has been determined in a continuous-flow experiment. The value obtained is 0.9 x 10⁶m^?1S^?1. Furthermore the dissociation rate constant is determined from the rate of the peroxidase-catalyzed oxidation of bilirubin in a bilirubin-albumin solution. This figure is 3.1 × 10^?2s ¹. Calculation of the apparent binding equilibrium constant from the two rate constants gives 2.9 x 10⁷m^?1. The above mentioned peroxidase oxidation has also been used for a direct estimation of the binding equilibrium constant giving 2.7 × 10⁷m^?1. All experiments are carried out at 36 °C and pH 7.4.     

Antibody-directed liposomes. Determination of affinity constants for soluble and liposome-bound antifluorescein

We have used the binding of liposomes conjugated with antifluorescein antibody specific for fluorescein isothiocyanate-modified erythrocytes as a model for multivalent antigen-antibody interactions. We examined a series of liposome preparations which were conjugated to between 0 and 332 active antibodies per liposome. The antigen binding capacity and mean intrinsic affinity of the soluble and conjugated antibody were determined by fluorescence quenching of carboxyfluorescein. Liposome-cell interaction data were fitted with a Scatchard-type equation. Functional affinity of liposomes for cells was up to 1000-fold greater than the intrinsic affinity of the antibody for soluble ligand. Analysis for binding at high cell concentrations revealed that liposome-induced cell agglutination reduces the number of available binding sites per cell.