4-nitroimidazole binding to horse metmyoglobin: evidence for preferential anion binding

The ionization of 4-nitroimidazole to 4-nitroimidazolate was investigated as a function of ionic strength. The apparent pKa varies from 8.99 to 9.50 between 0.001 and 1.0 M ionic strength, respectively, at 25 degrees C. The ionic strength dependence of this ionization is anomalous. The binding of 4-nitroimidazole by horse metmyoglobin was studied between pH 5.0 and 11.5 and as a function of ionic strength between 0.01 and 1.0 M. The association rate constant is pH-dependent, varying from 24 M(-1)s(-1) at pH 5 to a maximum value of 280 M(-1)s(-1) at pH 9.5 and then decreasing to 10 M(-1)s(-1) at pH 11.5 in 0.1 M ionic strength buffers. The dissociation rate constant has a much smaller pH dependence, varying from 0.082 s(-1) at low pH to 0.035 s(-1) at high pH, with an apparent pKa of 6.5. The binding affinity of 4-nitroimidazole to horse metmyoglobin is about 2.5 orders of magnitude stronger than that for imidazole and this increased affinity is attributed to the much slower dissociation rate for 4-nitroimidazole compared to that of imidazole. Although the ionic strength dependence of the binding rate is small and secondary kinetic salt effects can account for the ionic strength dependence of the association rate constant, the pH dependence of the rate constants and microscopic reversibility arguments indicate that the anionic form of the ligand binds more rapidly to all forms of metmyoglobin than does the neutral form of the ligand. However, the spectrum of the complex is similar to model complexes involving neutral imidazole and not imidazolate. The latter observation suggests that the initial metmyoglobin/4-nitroimidazolate complex rapidly binds a proton and the neutral form of the bound ligand is stabilized, probably through hydrogen binding with the distal histidine.     

Effect of pH on binding and dissociation of colony-stimulating factor

125I-labeled colony-stimulating factor (CSF) binds to granulocytic and monocytic cells in the bone marrow in an irreversible manner. Addition of a 1000-fold excess of unlabeled CSF does not displace the bound material. The present studies showed that brief exposures to pH 2.7-5.0 caused a marked release of the bound material. Such treatments were nontoxic to the marrow cells as judged by trypan blue dye exclusion, assay of colony-forming cells, and by analysis of rebinding of fresh 125I-CSF to the acid-treated cells. The CSF released from marrow cells by low pH revealed two peaks of radioactivity on SDS-acrylamide gel. The first peak (67,500 Da) corresponded to native CSF; a second peak of 53,500 Da was observed. Despite this apparent mild degradation of CSF, the released material showed greater binding to marrow and greater precipitation by anti-CSF than the native 125I-CSF. Further studies showed that acid treatment of marrow cells led to stabilization of the CSF receptors. Pretreatment at pH 4.0 led to retention of binding sites after conversion of marrow cultures to pH 7.5 and incubation at 22-37 degrees C. In contrast, cells that were not exposed to low pH lost receptors rapidly at these temperatures. The extent of preservation of the binding sites was related to the duration of acid exposure. These studies indicate that CSF is retained on the cell surface after binding at 0 degree C and that the CSF can be eluted by acid conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of maize actin-depolymerising factor with actin and phosphoinositides and its inhibition of plant phospholipase C.

We have previously reported the isolation of three Zea mays genes that encode actin-depolymerising factors/cofilins, a family of low molecular weight actin regulating proteins. In the present study, we have characterised one of these proteins, ZmADF3. We report that ZmADF3 binds G-actin with a 1:1 stoichiometry, and that the interaction with F-actin is pH-sensitive. ZmADF3 co-sediments mainly with F-actin at pH 6.0 and mainly with G-actin at pH 9.0. This response is more similar to that of vertebrate cofilin and ADF than to that of Acanthamoeba actophorin which, although more similar in primary sequence to ZmADF3, is not pH sensitive. However, ZmADF3 requires a more basic environment to depolymerise actin relative to either vertebrate ADF or cofilin. Filaments decorated with ZmADF3 at low pH are very rapidly depolymerised upon raising the pH, which is consistent with a severing mechanism for the disruption of actin filaments. Also, we demonstrate that ZmADF3 binds specific polyphosphatidylinositol lipids, especially phosphatidylinositol 4,5-bisphosphate (PIP₂), and we show that this binding inhibits the actin-depolymerising function of ZmADF3. Moreover, we show that a consequence of ZmADF3 binding PIP₂ is the inhibition of the activity of polyphosphatidylinositol specific plant phospholipase C, indicating the possibility of reciprocal modulation of this major signalling pathway and the actin cytoskeleton.     

Structural and biophysical studies of PCSK9 and its mutants linked to familial hypercholesterolemia

Proprotein convertase subtilisin kexin type 9 (PCSK9) lowers the abundance of surface low-density lipoprotein (LDL) receptor through an undefined mechanism. The structure of human PCSK9 shows the subtilisin-like catalytic site blocked by the prodomain in a noncovalent complex and inaccessible to exogenous ligands, and that the C-terminal domain has a novel fold. Biosensor studies show that PCSK9 binds the extracellular domain of LDL receptor with K(d) = 170 nM at the neutral pH of plasma, but with a K(d) as low as 1 nM at the acidic pH of endosomes. The D374Y gain-of-function mutant, associated with hypercholesterolemia and early-onset cardiovascular disease, binds the receptor 25 times more tightly than wild-type PCSK9 at neutral pH and remains exclusively in a high-affinity complex at the acidic pH. PCSK9 may diminish LDL receptors by a mechanism that requires direct binding but not necessarily receptor proteolysis.     

pH-dependent rate of formation of the gelsolin-actin complex from gelsolin and monomeric actin

The assembly of gelsolin with actin was followed by the increase of the fluorescence intensity of a fluorescence label bound to actin. The time course of the formation of the gelsolin-actin complex in the presence of micromolar [Ca2+] could be quantitatively interpreted by a model in which one actin molecule binds slowly to gelsolin in a rate-determining step and subsequently a second actin molecule is bound at least 40 times more rapidly. The rate of binding of the first actin molecule to gelsolin was found to be remarkably slow and to depend on the pH. The rate constants of formation of the gelsolin-actin complex range from 1.5 X 10(4) M-1 s-1 at pH 8 to 7 X 10(4) M-1 s-1 at pH 6.     

Kinetic consequences of a slow substrate binding step in group transferases. Interpretation of product inhibition experiments.

The steady state kinetic properties of a simple model for an enzyme catalyzed group transfer reaction between two substrates have been calculated. One substrate is assumed to bind slowly and the other rapidly to the enzyme. Apparent substrate inhibition or substrate activation by the rapidly binding substrate may result if the slowly binding substrate binds at unequal rates to the free enzyme and to the complex between the enzyme and the rapidly binding substrate. Competitive inhibition by each product with respect to its structurally analogous substrate is to be expected if both substrates are in rapid equilibrium with their enzyme-substrate complexes. This product inhibition pattern, however, may also be observed when one substrate binds slowly. Noncompetitive inhibition with respect to the rapidly binding substrate by its structurally analogous product may result if the slowly binding substrate binds more slowly to the enzyme-product complex than to the free enzyme. Inhibition by substrate analogs which are not products should follow the same rules as inhibition by products. Thus substrate analog inhibition experiments are not particularly informative. The form of inhibition by "transition state analog" inhibitors should reveal which substrate binds slowly. There is no sharp conceptual distinction between ordered and random "kinetic mechanisms". I therefore suggest that the use of these concepts should be abandoned.     

Binding of fluorescent lanthanides to rat liver mitochondrial membranes and calcium ion-binding proteins.

(1) Tb3+ binding to mitochondrial membranes can be monitored by enhanced ion fluorescence at 545 nm with excitation at 285 nm. At low protein concentrations (less than 30 mug/ml) no inner filter effects are observed. (2) This binding is localized at the external surface of the inner membrane and is unaffected by inhibitors of respiration or oxidative phosphorylation. (3) A soluble Ca2+ binding protein isolated according to Lehninger, A.L. ((1971) Biochem. Biophys. Res. Commun. 42, 312-317) also binds Tb3+ with enhanced ion fluorescence upon excitation at 285 nm. The excitation spectrum of the isolated protein and of the intact mitochondria are indicative of an aromatic amino acid at the cation binding site. (4) Further characterization of the Tb3+-protein interaction revealed that there is more than one binding site per protein molecule and that these sites are clustered (less than 20 A). Neuraminidase treatment or organic solvent extraction of the protein did not affect fluorescent Tb3+ binding. (5) pH dependency studies of Tb3+ binding to the isolated protein or intact mitochondria demonstrated the importance of an ionizable group of pK greater than 6. At pH less than 7.5 the amount of Tb3+ bound to the isolated protein decreased with increase in pH as monitored by Tb3+ fluorescence. With intact mitochondria the opposite occurred with a large increase in Tb3+ fluorescence at higher pH. This increase was not observed when the mitochondria were preincubated with antimycin A and rotenone.     

A pH-sensitive heparin-binding sequence from Baculovirus gp64 protein is important for binding to mammalian cells but not to Sf9 insect cells

Binding to heparan sulfate is essential for baculovirus transduction of mammalian cells. Our previous study shows that gp64, the major glycoprotein on the virus surface, binds to heparin in a pH-dependent way, with a stronger binding at pH 6.2 than at 7.4. Using fluorescently labeled peptides, we mapped the pH-dependent heparin-binding sequence of gp64 to a 22-amino-acid region between residues 271 and 292. Binding of this region to the cell surface was also pH dependent, and peptides containing this sequence could efficiently inhibit baculovirus transduction of mammalian cells at pH 6.2. When the heparin-binding peptide was immobilized onto the bead surface to mimic the high local concentration of gp64 on the virus surface, the peptide-coated magnetic beads could efficiently pull down cells expressing heparan sulfate but not cells pretreated with heparinase or cells not expressing heparan sulfate. Interestingly, although this heparin-binding function is essential for baculovirus transduction of mammalian cells, it is dispensable for infection of Sf9 insect cells. Virus infectivity on Sf9 cells was not reduced by the presence of heparin or the identified heparin-binding peptide, even though the peptide could bind to Sf9 cell surface and be efficiently internalized. Thus, our data suggest that, depending on the availability of the target molecules on the cell surface, baculoviruses can use two different methods, electrostatic interaction with heparan sulfate and more specific receptor binding, for cell attachment.     

Structural insights into the mechanism of pH-dependent ligand binding and release by the cation-dependent mannose 6-phosphate receptor

The cation-dependent mannose 6-phosphate receptor (CD-MPR) is a key component of the lysosomal enzyme targeting system that binds newly synthesized mannose 6-phosphate (Man-6-P)-containing acid hydrolases and transports them to endosomal compartments. The interaction between the MPRs and its ligands is pH-dependent; the homodimeric CD-MPR binds lysosomal enzymes optimally in the pH environment of the trans Golgi network (pH approximately 6.5) and releases its cargo in acidic endosomal compartments (相似文献   

Molten globule formation in apomyoglobin monitored by the fluorescent probe Nile Red

The interaction of nile red (NR) with apomyoglobin (ApoMb) in the native (pH 7) and molten globule (pH 4) states was investigated using experimental and computational methods. NR binds to hydrophobic locations in ApoMb with higher affinity (K(d) = 25 +/- 5 microM) in the native state than in the molten globule state (K(d) = 52 +/- 5 microM). In the molten globule state, NR is located in a more hydrophobic environment. The dye does not bind to the holoprotein, suggesting that the binding site is located at the heme pocket. In addition to monitoring steady-state properties, the fluorescence emission of NR is capable of tracking submillisecond, time-resolved structural rearrangements of the protein, induced by a nanosecond pH jump. Molecular dynamics simulations were run on ApoMb at neutral pH and at pH 4. The structure obtained for the molten globule state is consistent with the experimentally available structural data. The docking of NR with the crystal structure shows that the ligand binds into the binding pocket of the heme group, with an orientation bringing the planar ring system of NR to overlap with the position of two of the heme porphyrin rings in Mb. The docking of NR with the ApoMb structure at pH 4 shows that the dye binds to the heme pocket with a slightly less favorable binding energy, in keeping with the experimental K(d) value. Under these conditions, NR is positioned in a different orientation, reaching a more hydrophobic environment in agreement with the spectroscopic data.     

Rapid formation and high diffusibility of actin-cofilin cofilaments at low pH.

Cofilin is a small actin-binding protein that is known to bind both F-actin and G-actin, severing the former. The interaction of cofilin with actin is pH-sensitive, F-actin being preferentially bound at low pH and G-actin at higher pH, within the physiological range. Diffusion coefficients of F-actin with cofilin were measured by the fluorescence recovery after photobleaching (FRAP) technique. This has the potential for simultaneous and direct measurement of average polymer length via the average diffusion coefficient of the polymers (DLM) as well as the fraction of polymerized actin, fLM, present in solution. In the range of cofilin-actin ratios up to 1 : 1 and at both pH 6.5 and pH 8.0, the diffusion coefficients of the polymers increased with the amount of cofilin present in the complex, in a co-operative manner to a plateau. We interpret this as indicating co-operative binding/severing and that filaments less than a certain length cannot be severed further. Under the conditions used here, filaments were found to be more motile at pH 6.5 than at pH 8.0. At pH 8.0, some actin is expected to be sequestered as ADP-actin-cofilin complexes, with the remaining actin being present as long slowly diffusing filaments. At pH 6.5, however, cofilin binds to F-actin to form short rapidly diffusing cofilaments. These filaments form very rapidly from cofilin-actin monomeric complexes, possibly indicating that this complex is able to polymerize without dissociation. These findings may be relevant to the nuclear import of actin-cofilin complexes.     

Factors affecting the activity of bovicin HC5, a bacteriocin from Streptococcus bovis HC5: release, stability and binding to target bacteria

AIMS: To determine the factors affecting the release, stability and binding of bovicin HC5 to sensitive bacteria. METHODS AND RESULTS: Stationary phase Streptococcus bovis HC5 cultures had little cell-free bovicin HC5 activity until the final pH was <5.0, and even more bacteriocin was released by treatment with acidic NaCl (pH 2.0, 100 mmol l(-1)). Cultures grown with Tween 80 had more cell-free bovicin HC5 than untreated controls, but this nonionic detergent enhanced activity rather than release. Bovicin HC5 binding to S. bovis JB1 (a susceptible strain) was greater at pH values <6.0. Bovicin HC5 bound other sensitive Gram-positive bacteria, but not Gram-negative species. Cultures retained most of their activity for 35 days, but only if the final pH was <5.6. If the final pH was >5.6, peptidases destroyed much of the activity. CONCLUSIONS: Bovicin HC5 remains cell associated until the culture pH is <5.0, but it can be easily dissociated from the cell surface by acidic NaCl. It is highly stable in acidic environments and only binds sensitive bacteria at pH values <6.0. SIGNIFICANCE AND IMPACT OF THE STUDY: Streptococcus bovis HC5 does not have generally regarded as safe status. However, bovicin HC5 has a broad spectrum of activity and sensitive bacteria do not become resistant. Based on these results, bovicin HC5 may be a useful bacteriocin model.     

Characterization of [3H]-Valinomycin binding to red blood cell membrane

Valinomycin was tritiated by exchange and its biological activity found to be similar to that of nonlabeled drug. [³H]-valinomycin binds to red blood cell membranes following a biphasic pattern. High concentrations of the drug lead to an irreversible binding while low concentrations lead to a completely reversible binding. Maximum binding was obtained at acidic pH (pH 4.2) and physiological temperature (37°C). We demonstrate that valinomycin binds strongly to the lipidic phase of the membrane. When binding to erythrocytes and reticulocytes was compared, it was found that the mature red blood cells had less binding capacity than the reticulocytes.     

IgG transcytosis and recycling by FcRn expressed in MDCK cells reveals ligand-induced redistribution

The neonatal Fc receptor (FcRn) transports IgG across epithelial cells and recycles serum IgG. FcRn binds IgG at the acidic pH of endosomes and releases IgG at the basic pH of blood. We expressed rat FcRn in polarized MDCK cells and demonstrated that it functions in transcytosis and recycling of IgG. In the absence of IgG, FcRn is distributed predominantly apically, but redistributes to basolateral locations upon IgG addition, indicating that ligand binding induces a signal that stimulates transcytosis. FcRn transcytoses IgG more efficiently in the apical to basolateral than the reverse direction when IgG is internalized by receptor-mediated endocytosis at acidic pH or by fluid phase endocytosis at basic pH. The PI 3-kinase inhibitor wortmannin disrupts basolateral recycling and transcytosis in both directions, but only minimally reduces apical recycling. Confocal imaging and quantitative IgG transport studies demonstrate that apically-internalized IgG recycles to the apical surface mainly from wortmannin-insensitive apical early endosomes, whereas FcRn-IgG complexes that transcytose to the basolateral surface pass through downstream Rab11-positive apical recycling endosomes and transferrin-positive common endosomal compartments.     

A comparative study on two fungal lipases from Thermomyces lanuginosus and Yarrowia lipolytica shows the combined effects of detergents and pH on lipase adsorption and activity

The effects of various detergents and pH on the interfacial binding and activity of two fungal lipases from Yarrowia lipolytica (YLLIP2) and Thermomyces lanuginosus (TLL) were investigated using trioctanoin emulsions as well as monomolecular films spread at the air-water interface. Contrary to TLL, YLLIP2 was found to be more sensitive than TLL to interfacial denaturation but it was protected by detergent monomers and lowering the temperature. At pH 7.0, both the interfacial binding and the activities on trioctanoin of YLLIP2 and TLL were inhibited by sodium taurodeoxycholate (NaTDC). At pH 6.0, however, YLLIP2 remained active on trioctanoin in the presence of NaTDC, whereas TLL did not. YLLIP2 activity on trioctanoin was associated with strong interfacial binding of the enzyme to trioctanoin emulsion, whereas TLL was mostly detected in the water phase. The combined effects of bile salts and pH on lipase activity were therefore enzyme-dependent. YLLIP2 binds more strongly than TLL at oil-water interfaces at low pH when detergents are present. These findings are particularly important for lipase applications, in particular for enzyme replacement therapy in patients with pancreatic enzyme insufficiency since high detergent concentrations and highly variable pH values can be encountered in the GI tract.     

Interactions of diphtheria toxin B-fragment with cells. Role of amino- and carboxyl-terminal regions.

The B-fragment of diphtheria toxin binds to cell surface receptors and facilitates entry of the enzymatically active A-fragment into the cytosol. The roles of the amino- and carboxyl-terminal regions of the B-fragment in interactions with the cell membrane were studied by measuring specific binding, insertion into membranes at low pH, and formation of cation-selective channels, as well as by toxicity measurements after association with active A-fragment. Deletion of the amino-terminal 12 amino acids of the B-fragment did not affect its ability to bind to receptors and to form ion channels at low pH, whereas both abilities were strongly impaired when one more amino acid (Trp206) was removed. Replacement of the amino-terminal 31 residues with an amphipathic sequence from human apolipoprotein A1 restored receptor binding but not ion channel formation. The binding to cells was virtually abolished when 9 residues were deleted from the carboxyl terminus. Deletion of only 4 residues or extension by 12 residues did not prevent specific binding, but reduced insertion, channel formation, and toxicity. Those deletions that reduced receptor binding ability increased the trypsin sensitivity of the B-fragment. The results indicate that the amino- and carboxyl-terminal regions of diphtheria toxin B-fragment are important for receptor binding, possibly because they contribute to keep the B-fragment in a binding-competent conformation. Small alterations in the carboxyl-terminal end reduced insertion, channel formation, and toxicity more than the ability of the B-fragment to bind to cells.     

Binding site of Fe3+ at purine of ATP as studied by NMR

The binding site of Fe3+ in the purine base of adenosine 5'-triphosphate (ATP) was studied by nuclear magnetic resonance (NMR). The NMR relaxation rates (R1) of 1H and 31P in ATP solutions free of and containing ferric ions were measured in the pH range of 3-10. It was found that Fe3+ selectively enhanced the relaxation rate of protons. In the presence of Fe3+, the R1 of H2 was much bigger than that of H8 at a lower pH (3-4.5), while at a higher pH (5.5-7.5) the R1 of H8 was more enhanced than H2. At a pH of around 5, both H2 and H8, as well as all three phosphorous, showed a sudden jump in R1. When pH>8, Fe3+ failed to show appreciable enhancement of R1 to all protons and phosphorous. The quantitative data of relaxation rate enhancements suggest that the binding site of Fe3+ in ATP is strongly dependent on pH. At lower pH values, Fe3+ binds N1 but at higher pH it binds to N7. When pH is around 5, the whole purine base donates the aromatic pi-electrons to the ferric ion, forming a ferrocene-like complex, while when pH>8, ATP could not form complexes with Fe3+.     

NMR studies of the quaternary structure and heterogeneity of nitrosyl- and methemoglobin.

NMR was used to study the quaternary structure of nitrosyl- and methemoglobin, the kinetics and equilibrium behavior of nitric oxide binding, and the oxidation of hemoglobin. The -9.6 ppm (from H2O) resonance was used as a measure of nitrosylhemoglobin molecules in the T quaternary structure. We found that stripped nitrosylhemoglobin is 70% in the T state below pH 6.4, and is in the R state above. Inositol hexaphosphate (IHP) raises this transition point to pH 7.5. For stripped aquomethemoglobin, the T marker at -10 ppm is absent. In IHP, at pH 6.5 all of the molecules are in the T state. At both higher and lower pH they shift to the R state. The intensity decreases to half of its maximum at pH 5.5 and 7.4. The relative affinity of nitric oxide binding to the alpha and beta subunits was inferred from the intensities of the resonances at -12 and -18 ppm. Under conditions in which nitrosylhemoglobin exists in the T state, NO binds to the alpha subunit 10 times more strongly than it does to the beta subunit. The kinetic experiments reveal that it binds to the two subunits at the same rate and that it dissociates at 5 x 10(-3) s-1 from the beta subunit and at 5 x 10(-4) s-1 from alpha subunit. At high pH, the two subunits are ligated at the same rate. Potassium ferricyanide oxidation, at pH 6.0 in the absence of IHP, is about 3 times more favorable for the alpha than the beta subunit. Addition of IHP raises this preferential oxidation slightly. At pH 8.44, both alpha and beta subunits were oxidized at the same rate.     

pH in the endosome. Measurements during pinocytosis and receptor-mediated endocytosis

By fluorescence spectroscopy, the average pH within endocytic compartments was determined during endocytosis of fluorescein conjugates by macrophages and hepatocytes. In mouse macrophages and hepatocytes fluorescein conjugates taken up either in the fluid phase or by binding to cell surface receptors were rapidly transferred to an acidic compartment (pH 5-5.5). The half-time for this process was generally less than 4 min. The pH within yeast-containing phagosomes was also rapidly reduced to similar levels, following a unique and transient increase. In each case, the acid endosomal compartments involved probably do not contain lysosomal enzymes. When fluorescein conjugates of asialoglycoproteins were internalised by hepatocytes at 20 degrees C, no proteolysis occurred within the acidic endosome until the temperature was raised. Fluorescein conjugates of concanavalin A (conA) and polylysine were relatively more slowly internalised by macrophages. The half-times for uptake, estimated by fluorescence change, were comparable with the turnover time for bulk plasma membrane. The relatively high average pH experienced by these conjugates indicated that a small proportion of these non-specific cell-surface labels was always in contact with the extracellular medium.     

Kinetics of α-globin binding to α-hemoglobin stabilizing protein (AHSP) indicate preferential stabilization of hemichrome folding intermediate

Human α-hemoglobin stabilizing protein (AHSP) is a conserved mammalian erythroid protein that facilitates the production of Hemoglobin A by stabilizing free α-globin. AHSP rapidly binds to ferrous α with association (k'(AHSP)) and dissociation (k(AHSP)) rate constants of ≈10 μm(-1) s(-1) and 0.2 s(-1), respectively, at pH 7.4 at 22 °C. A small slow phase was observed when AHSP binds to excess ferrous αCO. This slow phase appears to be due to cis to trans prolyl isomerization of the Asp(29)-Pro(30) peptide bond in wild-type AHSP because it was absent when αCO was mixed with P30A and P30W AHSP, which are fixed in the trans conformation. This slow phase was also absent when met(Fe(3+))-α reacted with wild-type AHSP, suggesting that met-α is capable of rapidly binding to either Pro(30) conformer. Both wild-type and Pro(30)-substituted AHSPs drive the formation of a met-α hemichrome conformation following binding to either met- or oxy(Fe(2+))-α. The dissociation rate of the met-α·AHSP complex (k(AHSP) ≈ 0.002 s(-1)) is ～100-fold slower than that for ferrous α·AHSP complexes, resulting in a much higher affinity of AHSP for met-α. Thus, in vivo, AHSP acts as a molecular chaperone by rapidly binding and stabilizing met-α hemichrome folding intermediates. The low rate of met-α dissociation also allows AHSP to have a quality control function by kinetically trapping ferric α and preventing its incorporation into less stable mixed valence Hemoglobin A tetramers. Reduction of AHSP-bound met-α allows more rapid release to β subunits to form stable fully, reduced hemoglobin dimers and tetramers.