Kinetics of pyrophosphate induced iron release from diferric ovotransferrin

The kinetics of pyrophosphate-induced iron release from diferric ovotransferrin were studied spectrophotometrically at 37 degrees C in 0.1 M HEPES, pH 7.0. At high pyrophosphate concentrations, the kinetics are biphasic, indicating that the rates of iron release from the two, presumably noninteracting iron-binding sites of ovotransferrin are different. The pseudo-first-order rate constants for iron release from both the fast and slow sites exhibit a hyperbolic dependence on pyrophosphate concentrations. The data suggest that pyrophosphate forms complexes with the two iron-binding sites of ovotransferrin prior to iron removal. The stability constants of the complex formed with the fast site (Keqf) and slow site (Keqs) are 8.3 M-1 and 40.4 M-1, respectively. The first-order rate constants for the dissociation of ferric-pyrophosphate from the fast site (k2f) and the slow site (k2s) are 0.062 and 0.0044 min-1, respectively. Results from urea gel electrophoresis studies suggest that iron is released at a much faster rate from the N-terminal binding site of ovotransferrin. At high pyrophosphate concentration, only C-monoferric-ovotransferrin is detected during the course of iron release. At low pyrophosphate concentration, however, a detectable amount of N-monoferric-ovotransferrin is accumulated. This result is consistent with the kinetic finding that the site with a higher k2 (0.062 min-1) has a lower affinity toward pyrophosphate (Keq = 8.3 M-1) whereas the site with a lower k2 (0.0044 min-1) has a higher affinity for pyrophosphate (Keq = 40.4 M-1).     

Aluminum exchange between citrate and human serum transferrin and interaction with transferrin receptor 1

The kinetics and thermodynamics of Al(III) exchange between aluminum citrate (AlL) and human serum transferrin were investigated in the 7.2-8.9 pH range. The C-site of human serum apotransferrin in interaction with bicarbonate removes Al(III) from Al citrate with an exchange equilibrium constant K1 = (2.0 +/- 0.6) x 10(-2); a direct second-order rate constant k1 = 45 +/- 3 M(-1) x s(-1); and a reverse second-order rate constant k(-1) = (2.3 +/- 0.5) x 10(3) M(-1) x s(-1). The newly formed aluminum-protein complex loses a single proton with proton dissociation constant K1a = (15 +/- 3) nM to yield a first kinetic intermediate. This intermediate then undergoes a modification in its conformation followed by two proton losses; first-order rate constant k2 = (4.20 +/- 0.02) x 10(-2) s(-1) to produce a second kinetic intermediate, which in turn undergoes a last slow modification in the conformation to yield the aluminum-loaded transferrin in its final state. This last process rate-controls Al(III) uptake by the N-site of the protein and is independent of the experimental parameters with a constant reciprocal relaxation time tau3(-1) = (6 +/- 1) x 10(-5) x s(-1). The affinities involved in aluminum uptake by serum transferrins are about 10 orders of magnitude lower than those involved in the uptake of iron. The interactions of iron-loaded transferrins with transferrin receptor 1 occur with average dissociation constants of 3 +/- 1 and 5 +/- 1 nM for the only C-site iron-loaded and of 6.0 +/- 0.6 and 7 +/- 0.5 nM for the iron-saturated ST in the absence or presence of CHAPS, respectively. No interaction is detected between receptor 1 and aluminum-saturated or mixed C-site iron-loaded/N-site aluminum-loaded transferrin under the same conditions. The fact that aluminum can be solubilized by serum transferrin in biological fluids does not necessarily imply that its transfer from the blood stream to cytoplasm follows the receptor-mediated pathway of iron transport by transferrins.     

Different stability of N- and C-domain of diferric ovotransferrin in urea and application to the determination of iron distribution between the two domains

The study of guanidine-HCl or thermal denaturation of diferric ovotransferrin (Fe2Tf) has revealed a simultaneous unfolding of the two domains of the protein (Ikeda et al. (1985) FEBS Lett. 182, 305-309). In urea denaturation of Fe2Tf, however, two distinct steps of unfolding were observed in the urea concentration range from 4.5 to 9 M at pH 8.0 and 37 degrees C by measuring the residual iron-bound protein (absorbance at 465 nm) and the remaining folded structures (circular dichroism at 222 nm). From a study of urea denaturation of partially iron-saturated Tf whose iron preferentially occupied the N-domain, it was found that the first and the second steps of denaturation corresponded to those of the N-terminal (4.5-6 M urea) and C-terminal domains (over 7 M urea), respectively. The N-domain of Fe2Tf was selectively unfolded in 7 M urea and digested with trypsin to provide an iron-bound C-terminal fragment (42 kDa) in good yield (about 80% of theoretical). The kinetic analysis of the decrease in A465 of Fe2Tf in 9 M urea showed that the N-domain unfolded 3 x 10(2) times faster than the C-domain. With partially iron-saturated Tf, the decrease of A465 in 9 M urea also proceeded in a biphasic manner and the ratio, the decrement in A465 of the rapid phase/the decrement in A465 of the slow phase, gave the value of iron distribution as Fe at the N-site/Fe at the C-site.     

Anion-mediated iron release from transferrins. The kinetic and mechanistic model for N-lobe of ovotransferrin

Iron release process of ovotransferrin N-lobe (N-oTf) to anion/chelators has been resolved using kinetic and mechanistic approach. The iron release kinetics of N-oTf were measured at the endosomal pH of 5.6 with three different anions such as nitrilotriacetate, pyrophosphate, and sulfate using stopped flow spectrofluorimetric method, all yielding clear biphasic progress curves. The two observed rate constants and the corresponding amplitudes obtained from the double exponential curve fit to the biphasic curves varied depending on the type and concentration of anions. Several possible models for the iron release kinetic mechanism were examined on the basis of a newly introduced quantitative equation. Results from the curve fitting analyses were consistent with a dual pathway mechanism that includes the competitive iron release from two different protein states, namely, X and Y, with the respective first order rate constants of K(1) and K(2) (X, domain closed holo N-oTf; Y, anion induced different conformer of holo N-oTf). The reversible interconversions of X to Y and Y to X are driven by the second order rate constant k(3) and the first order rate constant K(4), respectively. The obtained rate constants were greatly variable for the three anions depending on the synergistic or nonsynergistic nature. In the light of the anion-binding sites of N-oTf located crystallographically, the compatible mechanistic model that includes competitive anion binding to the iron coordination sites and to a specific anion site is suggested for the dual pathway iron release mechanism.     

Copper complexes at N- and C-site of ovotransferrin: quantitative determination and visible absorption spectrum of each complex

Copper complexes at the two sites of ovotransferrin (TF) differed markedly in the rate of Cu release by EDTA. During the reaction, lambda max of the remaining Cu-Tf complex shifted to red side, while the difference spectrum of FenCu2-nTf vs. FenTf in which the N-site had been preferentially occupied with Fe had lambda max at blue side from that of Cu2Tf, 440 nm. From these results, the intrinsic spectrum for Cu-complex at each site was assigned: lambda max 450 nm for N- and 430 nm for C-site. The differences in the release rate and the spectrum can be used for the identification of the two domains of Tf and for the analysis of metal-binding behavior of each site.     

Transferrin, is a mixed chelate-protein ternary complex involved in the mechanism of iron uptake by serum-transferrin in vitro?

Iron uptake by transferrin from triacetohydroxamatoFe(III) (Fe(AHA)3) in the presence of bicarbonate has been investigated between pH 7 and 8.2. The protein transits from the opened apo- to the closed holoform by several steps with the accumulation of at least three kinetic intermediates. All these steps are accompanied by proton losses, probably occurring from the protein ligands and the side-chains involved in the interdomain H-bonding nets. The minor bihydroxamatoFe(III) species Fe(AHA)2 exchanges its iron with the C-site of apotransferrin in interaction with bicarbonate without detectable formation of any intermediate protein-iron-ligand mixed complex; direct second-order rate constant k1=4.15(+/-0.05)x10(7) M(-1) s(-1). The kinetic product loses a single proton and undergoes a modification in its conformation followed by the loss of two or three protons; first-order rate constant k2=3.25(+/-0.15) s(-1). This induces a new modification in the conformation; first-order rate constant k3=5.90(+/-0.30)x10(-2) s(-1). This new modification in conformation rate controls iron uptake by the N-site of the protein and is followed by a single proton loss; K3a=6.80 nM. Finally, the holoprotein or the monoferric transferrin in its thermodynamic equilibrated state is produced by a last modification in the conformation occurring in about 4000 seconds. But for the Fe(AHA)3 dissociation and the involvement of Fe(AHA)2 in the first step of iron uptake, this mechanism is identical to that reported for iron uptake from FeNAc3. This implies that the exchange of iron between a chelate and serum-transferrin occurs by a single general mechanism. The nature of the iron-providing chelate is only important for the first kinetic step of the exchange, which can be slowed to such an extent that it rate limits the exchange of iron.     

Microtubule assembly kinetics. Changes with solution conditions.

The assembly kinetics of microtubule protein are altered by ionic strength, temperature and Mg2+, but not by pH. High ionic strength (I0.2), low temperature (T less than 30 degrees C) and elevated Mg2+ (greater than or equal to 1.2 mM) induce a transition from biphasic to monophasic kinetics. Comparison of the activation energy obtained for the fast biphasic step at low ionic strength (I0.069) shows excellent agreement with the values obtained at high ionic strength, low temperature and elevated Mg2+. From this observation it can be implied that the tubulin-containing reactant of the fast biphasic event is also the species that elongates microtubules during monophasic assembly. Second-order rate constants for biphasic assembly are 3.82(+/- 0.72) x 10(7) M-1.s-1 and 5.19(+/- 1.25) x 10(6) M-1.s-1, and for monophasic assembly the rate constant is 2.12(+/- 0.56) x 10(7) M-1.s-1. The microtubule number concentration is constant during elongation of microtubules for biphasic and monophasic assembly.     

Transferrins, the mechanism of iron release by ovotransferrin.

Iron release from ovotransferrin in acidic media (3 < pH < 6) occurs in at least six kinetic steps. The first is a very fast (相似文献   

Regulation of cAMP-dissociation kinetics in lactating rat mammary gland

The cAMP-dissociation kinetics of rat mammary gland cytosols are dependent upon the temperature of cAMP association. Dissociation rates (measured at pH 6.5, 24 degrees C) were biphasic (k = 0.08-0.23 min-1 and k = 0.02 min-1) and monophasic (k-1 = 0.02 min-1) after 0 degrees C and 24 degrees C association, respectively. The temperature-dependent change from an initial fast rate to an initial slow rate was observed at all concentrations of cAMP tested from 1 to 1000 nM. When the slow-dissociating site was associated with non-radioactive 8-bromo-cAMP, the dissociation rates of [3H]-cAMP from the remaining dissociating site was slow (k = 0.02 min-1) and fast (k = 0.05 min-1) at 24 degrees C and 0 degrees C associating rate can be converted to the slow-dissociating rate by warming. When 0.2 M sodium thiocyanate was added to the association mixture at 24 degrees C, biphasic dissociation rates of k = 0.23 min-1 and k = 0.02 min-1 were observed, suggesting that the chaotropic salt blocks the interconversion of rates. The data are consistent with the model for cAMP-dependent protein kinase which exhibits two binding sites with different affinities. The type II enzyme from mammary gland cytosol exhibits in addition the phenomenon of temperature-dependent interconversion of the two binding affinities.     

Kinetics of the release of aluminum from human serum dialuminum transferrin to citrate

The kinetics of release of Al3+ from human serum dialuminum transferrin (Al2Tf) to citrate were investigated at 37 degrees C, pH 7.4, mu = 0.7 M, by difference UV spectrophotometry. The two metal-binding sites are not identical but behave in a kinetically similar manner to give apparent second-order rate constants of 0.60 and 0.38 M-1 s-1, respectively, for release of the first Al3+ from Al2Tf. The rate constants for release of the second metal ion from the monoaluminum transferrins are 0.27 and 0.12 M-1 s-1. The kinetic scheme for release of A13+ from Al2Tf is therefore similar to that for release of Fe3+ from Fe2Tf, but the rate of constants for metal ion release are between two and four orders of magnitude larger.     

A kinetic study of the photochemical inactivation of adenylate kinases of Mycobacterium marinum and bovine heart mitochondria

Using incident light energy of about 76 mW.cm-2 in a dye-sensitized photooxidation reaction, we have investigated the possible involvement of one or both of the histidine residues in the catalytic activity of adenylate kinase (ATP:AMP phosphotransferase) of Mycobacterium marinum. We have done this by investigating the kinetics of photochemical inactivation of the enzyme. At pH 7.4, the kinetics of photoinactivation are biphasic with two different pseudo-first-order rate constants. Adenosine 5'-pentaphospho 5'-adenosine (Ap5A), ATP and, to some extent, AMP, all gave protection to the enzyme from inactivation. Amino-acid analysis of the photoinactivated enzyme indicated the loss of the two histidine residues. This, and the fact that photoinactivation occurred faster at alkaline compared to acidic pH, indicated the involvement of the histidine residues in the catalytic activity. A mathematical model is developed which assumes that both histidine residues are required for maximal catalytic activity: one is located peripherally, is exposed, and therefore is readily photooxidized (pseudo-first-order rate constant, k1 = 1.3.10(-2)s-1), while the other is located at the active site, involved in substrate-binding and is shielded (pseudo-first-order rate constant, k2 = 2.9.10(-4)s-1). However, this shielded histidine could be exposed and made more accessible to photooxidation either by raising the pH above 10, or alternatively, by the addition of 8 M acetamide (or 6 M guanidine). Under these conditions, which apparently cause unfolding of the protein molecule, the kinetics of photoinactivation change from biphasic to monophasic, suggesting that both histidine residues are equally exposed and are photooxidized at the same rate. Unlike the enzyme from M. marinum, adenylate kinase from bovine heart mitochondria shows monophasic kinetics of photoinactivation at pH 7.4, suggesting that only one of the six histidine residues is essential for catalytic activity, or if more than one, then they all must be equally exposed. Further, ATP, AMP or Ap5A did not provide protection against photoinactivation, suggesting that the histidine residue(s) involved in the catalytic activity must remain exposed after the substrates bind at the active site of the mitochondrial enzyme.     

Mechanism of formation of the complex between transferrin and bismuth, and interaction with transferrin receptor 1

The kinetics and thermodynamics of Bi(III) exchange between bismuth mononitrilotriacetate (BiL) and human serum transferrin as well as those of the interaction between bismuth-loaded transferrin and transferrin receptor 1 (TFR) were investigated at pH 7.4-8.9. Bismuth is rapidly exchanged between BiL and the C-site of human serum apotransferrin in interaction with bicarbonate to yield an intermediate complex with an effective equilibrium constant K(1) of 6 +/- 4, a direct second-order rate constant k(1) of (2.45 +/- 0.20) x 10(5) M(-1) s(-1), and a reverse second-order rate constant k(-1) of (1.5 +/- 0.5) x 10(6) M(-1) s(-1). The intermediate complex loses a single proton with a proton dissociation constant K(1a) of 2.4 +/- 1 nM to yield a first kinetic product. This product then undergoes a modification in its conformation followed by two proton losses with a first-order rate constant k(2) = 25 +/- 1.5 s(-1) to produce a second kinetic intermediate, which in turn undergoes a last modification in the conformation to yield the bismuth-saturated transferrin in its final state. This last process rate-controls Bi(III) uptake by the N-site of the protein and is independent of the experimental parameters with a constant reciprocal relaxation time tau(3)(-1) of (3 +/- 1) x 10(-2) s(-1). The mechanism of bismuth uptake differs from that of iron and probably does not involve the same transition in conformation from open to closed upon iron uptake. The interaction of bismuth-loaded transferrin with TFR occurs in a single very fast kinetic step with a dissociation constant K(d) of 4 +/- 0.4 microM, a second-order rate constant k(d) of (2.2 +/- 1.5) x 10(8) M(-1) s(-1), and a first-order rate constant k(-d) of 900 +/- 400 s(-1). This mechanism is different from that observed with the ferric holotransferrin and implies that the interaction between TFR and bismuth-loaded transferrin probably takes place on the helical domain of the receptor which is specific for the C-site of transferrin and HFE. The relevance of bismuth incorporation by the transferrin receptor-mediated iron acquisition pathway is discussed.     

CAMP-dissociation kinetics in hormone-dependent and -independent rat mammary tumor cytosols

Cytosols from 7, 12-dimethylbenz (alpha) anthracene-induced rat mammary tumors which exhibit different hormone-responsiveness were compared with respect to their cAMP-dissociation kinetics. At 22 degree C, pH 4.5, 1 micrometer cAMP, hormone-dependent mammary tumors exhibited monophasic dissociation rates with a rate constant of k-1 = 0.06 min-1. In contrast, hormone-independent mammary tumors exhibited biphasic dissociation curves with rate constants of k-1 = 0.47 and k-2 = 0.06 min-1. The binding of cAMP was completely reversible; radio-labeled ligand was completely dissociated by 1mM nonradioactive cAMP; the binding protein could be reassociated to its original binding level after dextran-coated charcoal adsorption. The mammary cytosols exhibited specific binding for cAMP which could be displaced partially by cGMP but not by ATP, ADP, AMP, or adenosine. Receptor inactivation during the course of incubation was negligible. Both mammary tissue cytosols exhibited similar association rates at 22 degree C, pH 4.5, 1 micrometer cAMP (k+1 = 5-7 x 10(5)M-1 min-1). These data indicate that mammary tissues exhibit 2 cAMP dissociation rates. Hormone-dependent mammary tumors exhibit a dissociation constant of a high affinity binding site (k-1/k+1 = 0.07 micrometer) whereas hormone-independent mammary tumors exhibit dissociation constants of one high affinity (k-1/k+1 = 0.07 micrometer) and a second low affinity site (k-1/k+1 = 0.05 micrometer).     

Kinetic characterization of the pH-dependent oligomerization of R67 dihydrofolate reductase.

Protein-protein recognition results from the assembly of complementary surfaces on two molecules that form a stable, noncovalent, specific complex. Our interest was to describe kinetic aspects of the recognition in order to understand the subtle molecular mechanism of association. R67 dihydrofolate reductase (DHFR) provides an ideal model to investigate kinetic parameters of protein-protein association since it is a homotetramer resulting from the pH-dependent dimerization of homodimers. We took advantage of the presence of a tryptophan residue at the dimer-dimer interface to monitor pH-dependent oligomerization of R67 DHFR using stopped-flow fluorescence techniques. Except for pH near neutrality where dissociation exhibited biphasic kinetics, association and dissociation followed monophasic kinetics fitted on a two-state model. Apparent rate constants of association k(on) and dissociation k(off) were determined at various pHs and pointed to the key role of a histidine located at the dimer-dimer interface in the pH control of tetramerization. The values of the tetramer-dimer equilibrium dissociation constant were calculated from the ratio k(off) /k(on) and correlated well with those previously measured at equilibrium. The thermodynamic parameters and the activation energies of both the association and dissociation were determined and indicated that the association is enthalpy driven and suggested that the formation of four hydrogen bonds (one per monomer) is responsible for the thermodynamic stability of the tetramer. Detailed analysis of the biphasic kinetics led to an original model, in which protonation of the tetramer is the triggering event for the dissociation process while the association involves primarily the unprotonated dimers.     

Kinetics of the removal of ferric ion from transferrin by aminoalkylphosphonic acids

The kinetics of ion removal at 25 degrees C in 0.1 M Tris, pH 7.4 by a series of phosphonic acids have been evaluated. The initial rate of iron removal is first order in ferric-transferrin, but shows a hyperbolic dependence on the concentration of the phosphonate ligand. At high ligand concentrations the reaction is clearly biphasic, and the data are interpreted in terms of nonequivalent rate constants for iron removal from the two transferrin iron-binding sites. Rate constants for three phosphonic acid ligands are approximately 0.025 min-1 and approximately 0.007 min-1 for the faster and slower binding sites. The results are discussed in relation to the conformational change mechanism for iron removal from transferrin proposed by Coward et al. [21].     

Monomer-dimer equilibrium and oxygen binding properties of ferrous Vitreoscilla hemoglobin

The monomer-dimer equilibrium and the oxygen binding properties of ferrous recombinant Vitreoscilla hemoglobin (Vitreoscilla Hb) have been investigated. Sedimentation equilibrium data indicate that the ferrous deoxygenated and carbonylated derivatives display low values of equilibrium dimerization constants, 6 x 10(2) and 1 x 10(2) M(-1), respectively, at pH 7.0 and 10 degrees C. The behavior of the oxygenated species, as measured in sedimentation velocity experiments, is superimposable to that of the carbonylated derivative. The kinetics of O(2) combination, measured by laser photolysis at pH 7.0 and 20 degrees C, is characterized by a second-order rate constant of 2 x 10(8) M(-1) s(-1) whereas the kinetics of O(2) release at pH 7.0 is biphasic between 10 and 40 degrees C, becoming essentially monophasic below 10 degrees C. Values of the first-order rate constants (at 20 degrees C) and of the activation energies for the fast and slow phases of the Vitreoscilla Hb deoxygenation process are 4.2 s(-1) and 19.2 kcal mol(-1) and 0.15 s(-1) and 24.8 kcal mol(-1), respectively. Thus the biphasic kinetics of Vitreoscilla Hb deoxygenation is unrelated to the association state of the protein. The observed biphasic oxygen release may be accounted for by the presence of two different conformers in thermal equilibrium within the monomer. The two conformers may be assigned to a structure in which the heme-iron-bound ligand is stabilized by direct hydrogen bonding to TyrB10 and a structure in which such interaction is absent. The slow interconversion between the two conformers may reflect a very large conformational rearrangement in the disordered distal pocket segment connecting helices C and E.     

Comparisons of redox kinetics of methemerythrin and mu-sulfidomethemerythrin. Implications for interactions with cytochrome b5

We have examined the effects on redox kinetics of changing the reduction potential of the mu-oxo-bridged binuclear iron center in octameric hemerythrin (Hr) from Phascolopsis gouldii. The opportunity to examine such effects is provided by the availability of mu-sulfidomethemerythrin (mu-S2-metHr), whose [Fe(III),Fe(III)]met----[Fe(II),Fe(III)]semi-met reduction potential is approximately 200 mV higher than that of methemerythrin (metHr). We have used, as redox partners to Hr, a set of metal complexes and the heme proteins deoxymyoglobin (Mb) and cytochrome b5. The latter protein from P. gouldii is a presumed physiological redox partner of Hr. Similar kinetics at pH 8 in the presence or absence of the allosteric effector perchlorate suggest reduction of the iron atom closer to the outer surface of each subunit in the Hr octamer during the met----semi-met transformation. For all reducing agents, the experimentally determined ratio of second-order rate constants for reductions of mu-S2-metHr and metHr, k12(mu-S2-met)/k12(met), is close to the value of 40 predicted by the simple Marcus relation for "outer-sphere" electron transfer. For oxidations of (semi-met)RHr and mu-S2-semi-metHr, the predicted value of 40 for k12[(semi-met)R]/k12(mu-S2-semi-met) is closely approximated when Fe(CN)6(3-) is used as oxidant. The ionic strength dependence of the second-order rate constant suggests electrostatic interactions of opposite charges during reduction of metHr by P. gouldii cytochrome b5.(ABSTRACT TRUNCATED AT 250 WORDS)     

A new role for the transferrin receptor in the release of iron from transferrin

Iron removal by pyrophosphate from human serum diferric transferrin and the complex of transferrin with its receptor was studied in 0.05 M HEPES or MES buffers containing 0.1 M NaCl and 0.01 M CHAPS at 25 degrees C at pH 7.4, 6.4, and 5.6. At each pH, the concentration of pyrophosphate was adjusted to achieve rates of release amenable to study over a reasonable time course. Released iron was separated from protein-bound iron by poly(ethylene glycol) precipitation of aliquots drawn from the reaction mixture at various times during the course of a kinetic run. The amount of 59Fe label associated with the protein and pyrophosphate was determined from the radioactivity of precipitate and supernatant, respectively, in each aliquot. Iron removal of 0.05 M pyrophosphate at pH 7.4 from diferric transferrin bound to the receptor is considerably slower than that from free diferric transferrin, with observed pseudo-first-order rate constants of 0.020 and 0.191 min-1, respectively. For iron removal by 0.01 M pyrophosphate at pH 6.4, corresponding rate constants are 0.031 and 0.644 min-1. However, at pH 5.6, iron removal by 0.001 M pyrophosphate is faster from diferric transferrin bound to its receptor than from free transferrin (observed rate constants of 0.819 and 0.160 min-1, respectively). Thus, the transferrin receptor not only facilitates the removal of iron from diferric transferrin at the low pH that prevails in endocytic vesicles but may also reduce its accessibility to iron acceptors at extracellular pH, thereby minimizing the likelihood of nonspecific release of iron from transferrin at the cell surface.     

铁核结构对马脾铁蛋白释放铁动力学的影响

建立Ｈ^% 参与马脾铁蛋白释放铁的动力方程,Ｈ^ 以１／２级反应方式参与铁蛋白释放铁核表层的铁。在酸性介质（ＰＨ６．５）中,铁蛋白释放铁的总平均速率（３３２Ｆe^3 /HSF.min)比在碱性介质（Ｐ8Ｈ８．０）中放铁的总平均速率（７３Ｆe^3 /HSF.min)高４．６倍,铁蛋白的铁核结构和外加的磷酸盐均能影响该蛋白释放的速率,但并不改变其反应级数。     

Kinetics of electron entry, exit, and interflavin electron transfer during catalysis by sarcosine oxidase.

Sarcosine oxidase contains 1 mol of covalently bound plus 1 mol of noncovalently bound FAD per active site. The first phase of the anaerobic reduction of the enzyme with sarcosine converts oxidized enzyme to an equilibrium mixture of two-electron-reduced forms (EH2) and occurs at a rate (2700 min-1, pH 8.0) similar to that determined for the maximum rate of aerobic turnover in steady-state kinetic studies (2600 min-1). The second phase of the anaerobic half-reaction converts EH2 to the four-electron-reduced enzyme (EH4) and occurs at a rate (k = 350 min-1) which is 7-fold slower than aerobic turnover. Reaction of EH2 with oxygen is 1.7-fold faster (k = 4480 min-1) than aerobic turnover and 13-fold faster than the anaerobic conversion of EH2 to EH4. The results suggest that the enzyme cycles between fully oxidized and two-electron-reduced forms during turnover with sarcosine. The long wavelength absorbance observed for EH2 is attributable to a flavin biradical (FADH.FAD.-) which is generated in about 50% yield at pH 8.0 and in nearly quantitative yield at pH 7.0. The rate of biradical formation is determined by the rate of electron transfer from sarcosine to the noncovalent flavin since electron equilibration between the two flavins (k = 750 s-1 or 45,000 min-1, pH 8.0) is nearly 20-fold faster, as determined in pH-jump experiments. Only two of the three possible isoelectronic forms of EH2 are likely to transfer electrons to oxygen since the reaction is known to occur at the covalent flavin. However, equilibration among EH2 forms is probably maintained during reoxidation, consistent with the observed monophasic kinetics, since interflavin electron transfer is 10-fold faster than electron transfer to oxygen.