Kinetic measurements of Escherichia coli RNA polymerase association with bacteriophage T7 early promoters

During infection of Escherichia coli by bacteriophage T7, E. coli RNA polymerase utilizes only three promoters (A1, A2, and A3). In vitro, the A promoters predominate at very low polymerase concentration, but at higher polymerase concentration the minor B, C, D, and E promoters are used with equal efficiency. The binding constant for the initial association of polymerase with promoters and the forward rate of isomerization to an "open" complex capable of initiation have been measured for the A1, A3, C, and D promoters using the abortive initiation reaction. At 80 mM KCl, 37 degrees C, both major and minor promoters isomerize rapidly (t1/2 = 10 to 30 s). In contrast, initial binding to the minor promoters (KI = 10(7) ) is at least 10-fold weaker than binding to major promoters KI greater than or equal to 10(8) ), suggesting promoter selectivity in the T7 system occurs at the point of initial binding. Association kinetics of the A1 and C promoters on intact T7 were the same as measured on restriction fragments of length greater than or equal to 500 base pairs. All open complexes dissociated with half-lives longer than 1 h. Overall equilibrium binding constants estimated from kinetic measurements ranged from 10(10) to greater than or equal to 10(11) M-1 for minor and major promoters, respectively. Data on heparin attack and abortive initiation turnover rates indicate open complex polymerase conformation may be different at the A1 and A3 promoters.     

Penicillin-binding protein 2a from methicillin-resistant Staphylococcus aureus: kinetic characterization of its interactions with beta-lactams using electrospray mass spectrometry.

Penicillin-binding protein 2a (PBP2a) is the primary beta-lactam resistance determinant of methicillin-resistant Staphylococcus aureus (MRSA). MecA, the gene coding for PBP2a, was cloned with the membrane-anchoring region at the N-terminus deleted. The truncated protein (PBP2a) was overexpressed in Escherichia coli mostly in the soluble form accounting for approximately 25% of soluble cell protein and was purified to homogeneity. The purified protein was shown to covalently bind beta-lactams in an 1:1 ratio as determined by electrospray mass spectrometry. A novel method based on HPLC-elctrospray mass spectrometry has been developed to quantitatively determine the formation of the covalent adducts or acyl-PBP2a complexes. By using this method, combined with kinetic techniques including quench flow, we have extensively characterized the interactions between PBP2a and three beta-lactams and determined related kinetic parameters for the first time. The apparent first-order rate constants (ka) of PBP2a acylation by benzylpenicillin showed a hyperbolic dependence on the concentration of benzylpenicillin. This is consistent with the mechanism that the binding of the penicillin to PBP2a consists of reversible formation of a Michaelis complex followed by formation of the penicilloyl-PBP2a adduct, and allowed the determination of the individual kinetic parameters for these two steps, the dissociation constant Kd of 13.3 mM and the first-order rate constant k2 of 0.22 s-1. From these values, the second-order rate constant k2/Kd, the value reflecting the overall binding efficiency of a beta-lactam, of 16.5 M-1 s-1 was obtained. The fairly high Kd value indicates that benzylpenicillin fits rather poorly into the protein active site. Similar studies on the interaction between PBP2a and methicillin revealed k2 of 0.0083 s-1 and Kd of 16.9 mM, resulting in an even smaller k2/Kd value of 0.49 M-1 s-1. The rate constants k3 for deacylation of the acyl-PBP2a complexes, the third step in the interactions, were measured to be <1.5 x 10(-)5 s-1. These results indicate that the resistance of PBP2a to penicillin inactivation is mainly due to the extremely low penicillin acylating rate in addition to the low association affinity, but not to a fast rate of deacylation. Acylation of PBP2a by a high-affinity cephalosporin, Compound 1, also followed a saturation curve of ka versus the compound concentration, from which k2 = 0.39 s-1, Kd = 0.22 mM, and k2/Kd = 1750 M-1 s-1 were obtained. The 100-fold increase in the k2/Kd value as compared with that of benzylpenicillin is mostly attributable to the decreased (60-fold) Kd, indicating that the cephalosporin fits much better to the binding pocket of the protein.     

Effect of Escherichia coli initiation factors on the kinetics of N-Acphe-tRNAPhe binding to 30S ribosomal subunits. A fluorescence stopped-flow study

The mechanism of binding of N-AcPhe-tRNAPhe (yeast) to poly(U)-programmed Escherichia coli 30S ribosomal subunits and the effect of individual initiation factors (IF-1, IF-2, and IF-3) and GTP on this process have been studied by fluorescence stopped-flow kinetic measurements. The formation of the ternary complex was followed by an increase of both intensity and polarization of the fluorescence of a proflavin label located in the anticodon loop of the tRNA. The effect of the initiation factors and GTP is to increase the velocity of ternary complex formation (about 400-fold at 7 mM Mg2+). In the presence of the three initiation factors and GTP the formation of the ternary complex could be resolved into two partial reactions: a fast apparently second-order step (k12 = 5 x 10(6) M-1 s-1, k21 = 1.4 s-1) followed by a slow rearrangement step (k23 less than or equal to 0.1 s-1). The data suggest a mechanism in which the ternary complex is formed by at least two rearrangements of an initially formed preternary complex. The accelerating effects of both IF-2 and IF-3 can be understood by assuming a synergistic allosteric action of the factors on the 30S ribosomal subunit, whereas IF-1 appears to act indirectly by influencing the other two factors.     

1-Aminocyclopropanephosphonate: time-dependent inactivation of 1-aminocyclopropanecarboxylate deaminase and Bacillus stearothermophilus alanine racemase by slow dissociation behavior

1-Aminocyclopropanephosphonate (ACPP) was synthesized, and its effects on the pyridoxal 5'-phosphate linked enzymes 1-aminocyclopropanecarboxylate (ACPC) deaminase from Pseudomonas sp. ACPC and alanine racemase from Bacillus stearothermophilus were studied. ACPP was found to be a potent inhibitor of both enzymes with Km/Ki ratios of 500 and 2000, respectively. Inhibition for both enzymes was characterized by slow-binding (second-order rate constants less than 150 M-1 s-1) slow-dissociating behavior. Analysis of the pre-steady-state kinetics revealed a kinetically detectable intermediate E.I complex in the inhibition mechanism for the racemase but not for the deaminase. The one-step deaminase inhibition (Formula: see text) mechanism had an association rate constant (k1) of 100 M-1 s-1, a value 10(6)-fold slower than diffusion, suggesting either a slow alignment of the inhibitor at the enzyme active site or, more likely, the same mechanism as followed by racemase but with an E.I to E.I conversion rate (k3) that is sufficiently fast on the steady-state time scale so as to hinder detection of the initial weakly associated E.I intermediate. The E to E.I transition for the deaminase was further monitored by ultraviolet-visible and circular dichroism (CD) spectroscopies and found to exhibit a time-dependent shift in the visible absorption spectrum lambda max from 418 nm for the native enzyme to 333 nm at steady state, again consistent with a rapid E to E.I and slow E.I to E.I behavior. A rate constant for the absorbance shift of 150 M-1 s-1 was consistent with the k1 calculated in the inhibition studies.(ABSTRACT TRUNCATED AT 250 WORDS)     

The interaction between some serine proteinases and horse leucocyte inhibitor

Horse blood leucocyte cytosol exhibits a broad inhibitory activity against serine proteinases. The purified inhibitor was exposed to investigated enzymes (trypsin, chymotrypsin, elastases and serine proteinase from S. aureus) for variable time and the products were analyzed by gradient polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. The molar ratio I:E, association rate constants k on and inhibition constants Ki for the enzymes and inhibitor were determined. The examined elastases form stable, stoichiometric complexes with the inhibitor (Ki less than 10(-10) M), and do not undergo proteolytic degradation during 30 min incubation at 20 degrees C even at the 2-fold molar excess of the proteinases. The reactions with elastases are extremely rapid (k on greater than 10(7) M-1 s-1) and are completed within one second whereas similar reactions with chymotrypsin and trypsin are much slower (k on = 3 X 10(5) M-1 s-5 and 5 X 10(2) M-1 s-1, respectively). Serine proteinase from S. aureus neither react nor inactivates the investigated inhibitor. The complexes of the inhibitor with trypsin and chymotrypsin are digested even at a molar ratio I:E = 2:1. All these observations point out that the inhibitor from horse leucocyte cytosol is a specific and effective inhibitor of elastases.     

Limits of diffusion in the hydrolysis of substrates by the phosphotriesterase from Pseudomonas diminuta.

The catalytic mechanism for the enzymatic hydrolysis of a series of paraoxon analogues by the phosphotriesterase from Pseudomonas diminuta has been determined. The Br?nsted plots relating the pKa of the leaving group to the observed kinetic parameters, Vmax and V/Km, are both nonlinear. This observation is consistent with a change in the rate-limiting step from chemical to physical events as the pKa of the leaving group is decreased. This conclusion is confirmed by the effects of solvent viscosity on Vmax and V/Km for the same series of analogues. The data were fitted to the scheme E k1A in equilibrium k2 EA k3----EP k7----E'P k9----E + products where EA is the enzyme-substrate complex, EP is the enzyme-product complex, E'P is the enzyme-product complex after a viscosity-independent unimolecular reaction, and the values for k1, k2, k7, and k9 are 4.1 X 10(7) M-1 s-1, 2550 s-1, 3370 s-1, and 5940 s-1, respectively. The magnitude of the chemical step, represented by k3, is dependent on the pKa of the leaving group phenol as predicted by the Br?nsted equation (log k3 = beta pKa + C) where beta = -1.8 and the constant (C) = 17.7. The magnitude of beta indicates that the transition state for substrate hydrolysis is very product-like.     

The DNA guanyl radical: kinetics and mechanisms of generation and repair

The one-electron oxidation of DNA bases and single-stranded DNA was studied by pulse radiolysis of aqueous solutions from pH 7-7.4 at 20 degrees C. Thallic ions, Tl(II), were found to rapidly oxidize the purine nucleotides, deoxyguanosine 5'-monophosphate, k[Tl(II) + dGMP2-] = 3.4.10(9) M-1.s-1, and deoxyadenosine 5'-monophosphate, k[Tl(II) + dAMP2-] = 1.3.10(8) M-1.s-1. The reactivities of Tl(II) ions with model pyrimidine DNA bases, 1-methylcytosine and 1-methylthymine, were too low to be measured by pulse radiolysis, k less than 10(7) M-1.s-1. The Tl(II)-mediated oxidation of ssDNA, k = 2.8.10(8) M-1.s-1, produces DNA-guanyl radical, DNA-G.(-H), exclusively. The DNA-guanyl radical is found to be a potent oxidant in neutral media, E7 = 1.04 +/- 0.05 V. It rapidly oxidizes the aromatic amino acids in glycyl-tryptophan and tyrosine methyl ester, k = 3.6.10(7) M-1.s-1 and k = 1.7.10(8) M-1.s-1, respectively. These electron transfer processes indicate that a positive 'hole' may be transferred from DNA to a DNA-associated protein. The positive 'hole' in DNA can also be repaired by antioxidants, which are electron donors. The chemical repair of the DNA-guanyl radical by negatively charged antioxidants is slower than that by positively charged and neutral antioxidants.     

Interaction of RNA polymerase with promoters from bacteriophage fd.

Replicative form DNA of bacteriophage fd, which had been fragmented with the restriction endonuclease II from Hemophilus parainfluenzae (endo R- HpaII), was reacted with Escherichia coli RNA polymerase; the resulting stable preinitiation complexes were analysed using the filter binding assay followed by gel electrophoresis. At 120mM KCL the first-order rate constants for complex decay were determined to be 10(-2)-10(-6)s-1. The second-order rate constants for complex formation were found to be about 10(6) -10(7) M-1 s-1. From these values association constants for the individual promoters were calculated to be 2 x 10(-8) -2 x 10(-11) M-1. The rate of formation and the stability of promoter complexes was enhanced in superhelical DNA. No evidence was found for stable promoter-specific closed complexes consisting of enzyme and helical DNA. This and the kinetic data suggest that the unwinding of base pairs is already important early in promoter selection, and not only for the formation of the final open complex. The initiation of RNA synthesis form the preinitiation complex was faster than complex dissociation and complex formation for all promoters. Consequently, the initiation efficiency of a promoter is determined by the rate of complex formation, and not by its 'affinity' for the enzyme. No correlation was found between the relative order of the fd promoters for the binding and the dissociation reaction. This is explained by different structural determinants, for the two reactions, which are located in different parts of the promoter DNA.     

Structural determinants of the noncatalytic chain of tissue-type plasminogen activator that modulate its association rate with plasminogen activator inhibitor-1

In order to identify the regions of recombinant (r) tissue plasminogen activator (tPA) that mediate its kinetically relevant interaction with r-plasminogen activator inhibitor-1 (rPAI-1), we have determined the second-order association rate (k1) constants of domain-altered variants of tPA with rPAI-1, at 10 degrees C. With two-chain, wild-type recombinant tPA (tcwt-rtPA), obtained by expression of the human cDNA for tPA in five different cell systems (viz. insect cells, human kidney 293 cells, Chinese hamster ovary cells, human melanoma cells, and mouse C127 cells), the average k1 was 1.45 x 10(7) M-1 s-1 (range, 1.34 10(7) M-1 s-1-1.68 x 10(7) M-1 s-1). Since this value was not significantly different for the different tcwt-rtPA preparations, it appears as though the nature of the glycosylation of tPA plays little role in its initial interaction with PAI-1. The k1 determined for tcwt-rtPA was slightly higher than that of 0.87 x 10(7) M-1 s-1, obtained for a similar inhibition of human urokinase by rPAI-1. The k1 value obtained for single-chain (sc) wt-rtPA was approximately 6-fold lower than that of the two-chain molecules, results consistent with previous conclusions on this matter. The k1 value for tcwt-rtPA was not influenced by the presence of epsilon-aminocaproic acid, suggesting that the lysine-binding site associated with the kringle 2 (K2) region of tPA does not modulate the rate of its initial interaction with rPAI-1. Removal of the K2 domain from tPA, by recombinant DNA technology, results in a protein, F-E-K1-P (tc-r delta K2-tPA), containing only the finger (F), growth factor (E), kringle 1 (K1), and serine protease (P) domains. This variant protein was more rapidly inhibited by rPAI-1 (k1 = 3.00 x 10(7) M-1 s-1) than its wild-type counterparts. Deletion of both the K1 and K2 domains resulted in a variant molecule, F-E-P (tc-r delta K1 delta K2-tPA), that was slightly more rapidly inhibited by rPAI-1 (k1 = 2.01 x 10(7) M-1 s-1).(ABSTRACT TRUNCATED AT 400 WORDS)     

Heparin strongly decreases the rate of inhibition of neutrophil elastase by alpha 1-proteinase inhibitor

Heparin depresses the second-order rate constant ka for the inhibition of neutrophil elastase by alpha 1-proteinase inhibitor. High molecular mass heparin decreases ka from 1.3 x 10(7) M-1 s-1 to a limit of 4.6 x 10(4) M-1 s-1. Low molecular mass heparin is about 7-fold less effective. Dermatan sulfate and chondroitin sulfate are less efficient. Heparin preparations used in clinical care also strongly depress ka when tested at concentrations corresponding to their clinical efficacy. Heparin also decreases the ka for the elastase/eglin c and the cathepsin G/alpha 1-proteinase inhibitor systems but not that for the alpha 1-proteinase inhibitor/pancreatic elastase or trypsin pairs. These results, together with Sepharose-heparin binding studies, indicate that the ka-depressing effect of the polymer is related to its ability to form a tight complex with elastase but not with alpha 1-proteinase inhibitor. One mol of high molecular mass heparin binds 3 mol of neutrophil elastase with a Kd of 3.3 nM. Low molecular mass heparin binds elastase with a 1:1 stoichiometry and a Kd of 89 nM. For both heparins ka is lowest when elastase is fully saturated with heparin. From this we conclude that heparin decreases ka, because the heparin-elastase complex is able to slowly react with alpha 1-proteinase inhibitor and not because the inhibitor slowly dissociates the heparin-elastase complex. These findings may have important pathophysiological bearing.     

Steady-state kinetics of thiocyanate oxidation catalyzed by human salivary peroxidase

A steady-state kinetic analysis was made of thiocyanate (SCN-) oxidation catalyzed by human peroxidase (SPO) isolated from parotid saliva. For comparative purposes, bovine lactoperoxidase (LPO) was also studied. Both enzymes followed the classical Theorell-Chance mechanism under the initial conditions [H2O2] less than 0.2mM, [SCN-] less than 10mM, and pH greater than 6.0. The pH-independent rate constants (k1) for the formation of compound I were estimated to be 8 X 10(6) M-1 s-1 (SD = 1, n = 18) for LPO and 5 X 10(6) M-1 s-1 (SD = 1, n = 11) for SPO. The pH-independent second-order rate constants (k4) for the oxidation of thiocyanate by compound I were estimated to be 5 X 10(6) M-1 s-1 (SD = 1, n = 18) for LPO and 9 X 10(6) M-1 s-1 (SD = 2, n = 11) for SPO. Both enzymes were inhibited by SCN- at pH less than 6. The pH-independent equilibrium constant (Ki) for the formation of the inhibited enzyme-SCN- complex was estimated to be 24 M-1 (SD = 12, n = 8) for LPO and 44 M-1 (SD = 4, n = 10) for SPO. An apparent pH dependence of the estimated values for k4 and Ki for both LPO and SPO was consistent with a mechanism based on assumptions that protonation of compound I was necessary for the SCN- peroxidation step, that a second protonation of compound I gave an inactive form, and that the inhibited enzyme-SCN- complex could be further protonated to give another inactive form.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reduction kinetics of the ferredoxin-ferredoxin-NADP+ reductase complex: a laser flash photolysis study

The kinetics of reduction of spinach ferredoxin (Fd), ferredoxin-NADP+ reductase (FNR), and the Fd-FNR complex have been investigated by the laser flash photolysis technique. 5-Deazariboflavin semiquinone (5-dRf), generated in situ by laser flash photolysis under anaerobic conditions, rapidly reduced both oxidized Fd (Fdox) (k = 2 X 10(8) M-1 s-1) and oxidized FNR (FNRox) (K = 6.3 X 10(8) M-1 s-1) at low ionic strength (10 mM) at pH 7.0, leading to the formation of reduced Fd (Fdred) and FNR semiquinone (FNR.), respectively. At higher ionic strengths (310 and 460 mM), the rate constant for the reduction of the free Fdox increased about 3-fold (k = 6.7 X 10(8) M-1 s-1 at 310 mM and 6.4 X 10(8) M-1 s-1 at 460 mM). No change in the second-order rate constant for reduction of the free FNRox was observed at high ionic strength. At low ionic strength (10 mM), 5-dRf. reacted only with the FAD center of the preformed 1:1 Fdox-FNRox complex (k = 5.6 X 10(8) M-1 s-1), leading to the formation of FNR.. No direct reduction of Fdox in the complex was observed. No change in the kinetics occurred in the presence of excess NADP+. The second-order rate constant for reduction of Fdox by 5-dRf. in the presence of a stoichiometric amount of fully reduced FNR at low ionic strength was 7 X 10(6) M-1 s-1, i.e., about one-thirtieth the rate constant for reduction of free Fdox.(ABSTRACT TRUNCATED AT 250 WORDS)     

An elastase-dependent pathway of plasminogen activation

In reaction mixtures containing Glu-plasminogen, alpha 2-antiplasmin, and tissue plasminogen activator or urokinase, either pancreatic or leukocyte elastase enhances the rate of plasminogen activation by 2 or more orders of magnitude. This effect is the consequence of several reactions. (a) In concentrations on the order of 100 nM, elastase degrades plasminogen within 10 min to yield des-kringle1-4-plasminogen (mini-plasminogen), which is 10-fold more efficient than Glu-plasminogen as a substrate for plasminogen activators. Des-kringle1-4-plasminogen is insensitive to cofactor activities of fibrin(ogen) fragments or an endothelial cell cofactor. (b) Des-kringle1-4-plasmin is one-tenth as sensitive as plasmin to inhibition by alpha 2-antiplasmin: k" = 10(6) M-1 s-1 versus 10(7) M-1 s-1. (c) alpha 2-Antiplasmin is disabled efficiently by elastase, with a k" of 20,000 M-1 s-1. The elastase-dependent reactions are not influenced by 6-aminohexanoate. In diluted (10-fold) blood plasma, the capacity of endogenous inhibitors to block plasmin expression is suppressed by 30 microM elastase. It is proposed that elastases provide an alternative pathway for Glu-plasminogen activation and a mechanism for controlling initiation of fibrinolysis by urokinase-type plasminogen activators.     

Protease specificity and heparin binding and activation of recombinant protease nexin I

Structural and functional properties of alpha-protease nexin I (alpha-PNI) expressed in Chinese hamster ovary cells were studied. All three cysteines were in the reduced form, showing that the potential disulfide bridge between residues Cys117 and Cys131 was not formed. Heparin association rate enhancements were from ka = 8.3 x 10(5) to 0.7-1.6 x 10(9) M-1 s-1 for the interaction of PNI with thrombin, from ka = 5.1 x 10(3) to 3.5 x 10(5) M-1 s-1 for interaction with Factor Xa, and from ka = 2.2 x 10(6) to 1.0 x 10(7) M-1 s-1 for interaction with trypsin; there was no rate enhancement of the plasmin interaction (ka = 1.0 x 10(5) M-1 s-1). The minimal heparin pentasaccharide had no effect on these interactions. Cleavage of the reactive center loop of PNI by three different proteases gave the typical stressed to relaxed change in thermal stability, but unlike with antithrombin III, there was no loss of heparin affinity. A similar difference from antithrombin was that PNI-thrombin complexes retained normal heparin affinity. These results are compatible with a role for protease nexin I as a cell-associated thrombin inhibitor that remains bound to the cell surface even after complexing with the protease, as compared with the role of antithrombin III as a circulating inhibitor of thrombin that becomes activated on binding to the microvasculature and is released on complex formation.     

Comparison of the peroxidase reaction kinetics of prostaglandin H synthase-1 and -2.

Prostaglandin H synthase isoforms 1 and 2 (PGHS-1 and -2) each have a peroxidase activity and also a cyclooxygenase activity that requires initiation by hydroperoxide. The hydroperoxide initiator requirement for PGHS-2 cyclooxygenase is about 10-fold lower than for PGHS-1 cyclooxygenase, and this difference may contribute to the distinct control of cellular prostanoid synthesis by the two isoforms. We compared the kinetics of the initial peroxidase steps in PGHS-1 and -2 to quantify mechanistic differences between the isoforms that might contribute to the difference in cyclooxygenase initiation efficiency. The kinetics of formation of Intermediate I (an Fe(IV) species with a porphyrin free radical) and Intermediate II (an Fe(IV) species with a tyrosyl free radical, thought to be the crucial oxidant in cyclooxygenase catalysis) were monitored at 4 degrees c by stopped flow spectrophotometry with several hydroperoxides as substrate. With 15-hydroperoxyeicosatetraenoic acid, the rate constant for Intermediate I formation (k1) was 2.3 x 10(7) M-1 s-1 for PGHS-1 and 2.5 x 10(7) M-1 s-1 for PGHS-2, indicating that the isoforms have similar initial reactivity with this lipid hydroperoxide. For PGHS-1, the rate of conversion of Intermediate I to Intermediate II (k2) became the limiting factor when the hydroperoxide level was increased, indicating a rate constant of 10(2)-10(3) s-1 for the generation of the active cyclooxygenase species. For PGHS-2, however, the transition between Intermediates I and II was not rate-limiting even at the highest hydroperoxide concentrations tested, indicating that the k2 value for PGHS-2 was much greater than that for PGHS-1. Computer modelling predicted that faster formation of the active cyclooxygenase species (Intermediate II) or increased stability of the active species increases the resistance of the cyclooxygenase to inhibition by the intracellular hydroperoxide scavenger, glutathione peroxidase. Kinetic differences between the PGHS isoforms in forming or stabilizing the active cyclooxygenase species can thus contribute to the difference in the regulation of their cellular activities.     

Kinetics and mechanism of bilirubin binding to human serum albumin

The kinetics of bilirubin binding to human serum albumin at pH 7.40, 4 degrees C, was studied by monitoring changes in bilirubin absorbance. The time course of the absorbance change at 380 nm was complex: at least three kinetic events were detected including the bimolecular association (k1 = 3.8 +/- 2.0 X 10(7) M-1 S-1) and two relaxation steps (52 = 40.2 +/- 9.4 s-1 and k3 = 3.8 +/- 0.5 s-1). The presence of the two slow relaxations was confirmed under pseudo-first order conditions with excess albumin. Curve-fitting procedures allowed the assignment of absorption coefficients to the intermediate species. When the bilirubin-albumin binding kinetics was observed at 420 nm, only the two relaxations were seen; apparently the second order association step was isosbestic at this wavelength. The rate of albumin-bound bilirubin dissociation was measured by mixing the pre-equilibrated human albumin-bilirubin complex with bovine albumin. The rate constant for bilirubin dissociation measured at 485 nm was k-3 = 0.01 s-1 at 4 degrees C. A minimum value of the equilibrium constant for bilirubin binding to human albumin determined from the ratio k1/k-3 is therefore approximately 4 X 10(9) M-1.     

Kinetic studies on the interaction of eglin c with human leukocyte elastase and cathepsin G

We have investigated the inhibition of human leukocyte elastase and cathepsin G by recombinant Eglin c under near physiological conditions. The association rate constants k on of Eglin c for elastase and cathepsin G were 1.3 X 10(7) M-1 s-1 and 2 X 10(6) M-1 s-1, respectively. Under identical conditions, the k on for the association of human plasma alpha 1-proteinase inhibitor with the two leukocproteinases were 2.4 X 10(7) M-1 s-1 and 10(6) M-1 s-1, respectively. The consistency of these data could be verified using a set of competition experiments. The elastase-Eglin c interaction was studied in greater detail. The dissociation rate constant k off was determined by trapping of free elastase from an equilibrium mixture of elastase and Eglin c with alpha 1-proteinase inhibitor or alpha 2-macroglobulin. The rate of dissociation was very low (k off = 3.5 X 10(-5) s-1). The calculated equilibrium dissociation constant of the complex, Ki(calc) = k off/k on, was found to be 2.7 X 10(-12) M. Ki was also measured by adding elastase to mixtures of Eglin c and substrate and determining the steady-state rates of substrate hydrolysis. The Ki determined from these experiments (7.5 X 10(-11) M) was significantly higher than Ki(calc). This discrepancy might be explained by assuming that the interaction of Eglin c with elastase involves two steps: a fast binding reaction followed by a slow isomerization step. From the above kinetic constants it may be inferred that at a therapeutic concentration of 5 X 10(-7) M, Eglin c will inhibit leukocyte elastase in one second and will bind this enzyme in a "pseudo-irreversible" manner.