Dissociation Of Tetramer Research Articles

22] Hemoglobin-oxygen equilibrium binding: Rapid-scanning spectrophotometry and singular value decomposition

The knowledge of the interaction between oxygen (O2) and hemoglobin (Hb) is essential both for understanding oxygen transport phenomena and for testing theories of protein structure and function. The equilibrium reaction is measured routinely simply to estimate the position and shape of the O2-binding curve from, respectively, the O2 pressure at half saturation (P50) and the index of cooperativity (i.e., the Hill number, n), but thermodynamic analysis of individual binding steps is more difficult because of inherent complexities in the reaction. The Hb conformational transition is another area under study. Strong allosteric effectors, such as inositol hexaphosphate and bezafibrate, are being used to test this by possibly enhancing the signal. In the future, mathematical models will include calculations of tetramer dissociation and subunit differences in ligand binding. As instruments and theory improve, the critical components of the reaction will be resolved better and, once limiting assumptions are no longer required for experimental analyses, the evaluation of Hb-O2 equilibrium binding will become a simpler and more reliable process.

29] Measuring relative rates of hemoglobin oxidation and denaturation

Publisher Summary In aqueous media, without the active methemoglobin reductase system present in intact red blood cells, the ferrous heme groups of hemoglobin can auto-oxidize to form continually increasing amounts of high-spin ferric heme. Under conditions that promote tetramer dissociation, subsequent tertiary structural distortions in the globin monomers occur; these distortions are associated with iron spin-state transitions and possible geometric distortions of the heme moiety within individual heme pockets. Binding of low-spin ferric heme at the sixth coordinate position of the heme iron to normally distant amino acid side chains within the heme pocket can then result in the formation and accumulation of soluble and insoluble hemichromes. This chapter presents a generalized outline of this pathway. An alternate degradative path is also shown, wherein the high-spin ferric heme of hemoglobin is transferred to human serum albumin to form methemalbumin. The apoprotein thus formed is insoluble.

The existence of multiple tetrameric conformers of chicken liver pyruvate carboxylase and their roles in dilution inactivation.

The time-dependent loss of enzymic activity and tetrameric structure of chicken liver pyruvate carboxylase (EC 6.4.1.1) after dilution below 2 units/ml was apparently monophasic and first-order. When examined over a range of initial enzyme concentrations, both activity and tetrameric structure decayed to equilibrium levels which were dependent on the initial concentration. The observed rate constants for the loss of enzymic activity (i) showed no apparent dependence on the initial enzyme concentration, and (ii) were of similar magnitude to the corresponding rate constants of dissociation. Computer simulations of the most likely kinetic model suggest that the predominant form of the dissociated enzyme is the monomer. Dilution of pyruvate carboxylase in the presence of the allosteric activator acetyl-CoA largely prevented the subsequent dissociation of the tetrameric molecule. In addition, acetyl-CoA was able to cause a degree of activation and reassociation when added after dilution inactivation had been allowed to occur. Electron-microscopic observation showed the treatment with avidin before dilution markedly decreased the degree of dissociation of the enzyme tetramer. This structure-stabilizing effect of avidin was dependent on preincubation of the concentrated enzyme solution with acetyl-CoA. We propose that, over a range of protein concentrations, the tetrameric enzyme exists in two forms that are in equilibrium, and that acetyl-CoA alters the equilibrium to favour the more compact form.

Dependence of the permanent deformation of red blood cell membranes on spectrin dimer-tetramer equilibrium: implication for permanent membrane deformation of irreversibly sickled cells

Red blood cells (RBCs) in sickle cell anemia, transformed into a sickled shape by prolonged deoxygenation, or normal RBCs deformed by a prolonged micropipette aspiration become permanently stabilized in their abnormal shape. This semisolid plastic behavior is thought to involve an irreversible reorganization of the membrane skeleton, but the exact nature of this skeletal rearrangement is not known. In this study, we first asked whether the irreversible deformation is associated with a permanent stretching of the skeletal network, and then whether it is due to a rearrangement of skeletal components involving a disruption of pre-existing protein associations and the subsequent reassociation of new protein contacts. Having found no ultrastructural evidence of stretching of the skeletal lattice in membranes derived from permanently deformed RBCs, we addressed the possibility of reorganization of the proteins of the membrane skeleton. We examined the temperature dependence of irreversible cell deformation to see if it correlated with the known temperature dependence of spectrin tetramers to dimer dissociation and reassociation. Testing the shape irreversibility of both deoxygenated reversibly sickled cells and Nucleopore-aspirated normal cells, we found that both types of cells became permanently deformed when the prolonged incubation of applied force or deoxygenation was performed at 37 degrees C, the temperature at which spectrin tetramers were free to dissociate and reassociate. In contrast, both types of cells were able to regain their original discocytic shape if the prolonged incubation was performed at the lower temperature: at less than 13 degrees C instead of 37 degrees C. Furthermore, normal RBCs were incubated with inosine and pyruvate to elevate intracellular 2,3-diphosphoglycerate, the polyanion shown to destabilize spectrin-actin-protein 4.1 association. This did not result in a promotion of irreversible deformation of these cells. We conclude that the irreversible cell deformation observed at physiologic temperature is associated with a skeletal rearrangement through dissociation of spectrin tetramers to dimers and a subsequent reassociation of dimers to tetramers in the new (deformed) configuration. These findings may explain a permanent stabilization of irreversibly sickled cells in their abnormal shape in vivo.

Dependence of the Permanent Deformation of Red Blood Cell Membranes on Spectrin Dimer–Tetramer Equilibrium: Implication for Permanent Membrane Deformation of Irreversibly Sickled Cells

AbstractRed blood cells (RBCs) in sickle cell anemia, transformed into a sickled shape by prolonged deoxygenation, or normal RBCs deformed by a prolonged micropipette aspiration become permanently stabilized in their abnormal shape. This semisolid plastic behavior is thought to involve an irreversible reorganization of the membrane skeleton, but the exact nature of this skeletal rearrangement is not known. In this study, we first asked whether the irreversible deformation is associated with a permanent stretching of the skeletal network, and then whether it is due to a rearrangement of skeletal components involving a disruption of pre-existing protein associations and the subsequent reassociation of new protein contacts. Having found no ultrastructural evidence of stretching of the skeletal lattice in membranes derived from permanently deformed RBCs, we addressed the possibility of reorganization of the proteins of the membrane skeleton. We examined the temperature dependence of irreversible cell deformation to see if it correlated with the known temperature dependence of spectrin tetramers to dimer dissociation and reassociation. Testing the shape irreversibility of both deoxygenated reversibly sickled cells and Nuclepore-aspirated normal cells, we found that both types of cells became permanently deformed when the prolonged incubation of applied force or deoxygenation was performed at 37°C, the temperature at which spectrin tetramers were free to dissociate and reassociate. In contrast, both types of cells were able to regain their original discocytic shape if the prolonged incubation was performed at the lower temperature: at less than 13°C instead of 37°C. Furthermore, normal RBCs were incubated with inosine and pyruvate to elevate intracellular 2,3-diphosphoglycerate, the polyanion shown to destabilize spectrin–actin–protein 4.1 association. This did not result in a promotion of irreversible deformation of these cells. We conclude that the irreversible cell deformation observed at physiologic temperature is associated with a skeletal rearrangement through dissociation of spectrin tetramers to dimers and a subsequent reassociation of dimers to tetramers in the new (deformed) configuration. These findings may explain a permanent stabilization of irreversibly sickled cells in their abnormal shape in vivo.

Correlation of cofactor binding and the quaternary structure of pyruvate decarboxylase as revealed by 31P NMR spectroscopy

The pH dependence of the quaternary structure of pyruvate decarboxylase (EC 4.1.1.1) has recently been discovered [(1990) FEBS Lett. 266, 17–20; (1992) Biochemistry (in press)]. In the present study we have investigated the change in quaternary structure by observing the binding of the cofactor, thiamine pyrophosphate, using 31P NMR spectroscopy. The dissociation of the native tetramers into dimers when increasing the pH coincides with a weaker binding of the cofactor and loss of enzyme activity. The results provide further evidence that thiamine pyrophosphate is bound primarily via the β-phosphate moiety. In addition, a phosphoserine has been discovered in two of the four subunits.

Inactivation of Maize Leaf Phosphoenolpyruvate Carboxylase by the Binding to Chloroplast Membranes

Phosphoenolpyruvate carboxylase (PEPC) purified from maize (Zea mays L.) leaves associates with maize leaf chloroplast membrane in vitro. The binding of PEPC to the membrane results in enzyme inactivation. A protein isolated from a maize leaf chloroplast membrane preparation inactivated PEPC. Treatment with membrane preparation or with partially purified inactivating protein accelerates PEPC inactivation at low temperature (4 degrees C). Interaction of PEPC with chloroplast membrane or inactivating protein may inactivate the enzyme by influencing dissociation of the enzyme active tetramer.

Unfolding of trp repressor studied using fluorescence spectroscopic techniques.

The unfolding properties of the trp repressor of Escherichia coli have been studied using a number of different time-resolved and steady-state fluorescence approaches. Denaturation by urea was monitored by the average fluorescence emission energy of the intrinsic tryptophan residues of the repressor. These data were consistent with a two-state transition from dimer to unfolded monomer with a free energy of unfolding of 19.2 kcal/mol. The frequency response profiles of the fluorescence emission brought to light subtle urea-induced modifications of the intrinsic tryptophan decay parameters both preceding and following the main unfolding transition. The increase of lifetime induced by urea required higher concentrations of urea than the increase in the total intensity described by Gittelman and Matthews [(1990) Biochemistry 29, 7011]. This indicates that the intensity increase has both dynamic and static origins. To assess the effect of tryptophan binding upon repressor stability, and to determine whether repressor oligomerization would be detectable in an unfolding experiment, we examined denaturation profiles of repressor labeled with the long-lived fluorescence probe 5-(dimethylamino)naphthalene-1-sulfonyl (DNS), by monitoring the average rotational correlation time of the probe. These experiments revealed a protein concentration dependent transition at low urea concentrations. This transition was promoted by tryptophan binding. We ascribe this transition to urea-induced dissociation of repressor tetramers. The main unfolding transition of the dimer to unfolded monomer was also observable using this technique, and the free energies associated with this transition were 18.3 kcal/mol in the absence of tryptophan and 24.1 kcal/mol in its presence, demonstrating that co-repressor binding stabilizes the repressor dimer against denaturation.(ABSTRACT TRUNCATED AT 250 WORDS)

Amphiphilic and nonamphiphilic forms of bovine and human dopamine beta-hydroxylase.

We show that human and bovine dopamine beta-hydroxylases (DBH) exist under three main molecular forms: a soluble nonamphiphilic form and two amphiphilic forms. Sedimentation in sucrose gradients and electrophoresis under nondenaturing conditions, by comparison with acetylcholinesterase (AChE), suggest that the three forms are tetramers of the DBH catalytic subunit and bind either no detergent, one detergent micelle, or two detergent micelles. By analogy with the Gna4 and Ga4 AChE forms, we propose to call the nonamphiphilic tetramer Dna4 and the amphiphilic tetramers Da4I and Da4II. In addition to the major tetrameric forms, DBH dimers occur as very minor species, both amphiphilic and nonamphiphilic. Reduction under nondenaturing conditions leads to a partial dissociation of tetramers into dimers, retaining their amphiphilic character. This suggests that the hydrophobic domain is not linked to the subunits through disulfide bonds. The two amphiphilic tetramers are insensitive to phosphatidylinositol phospholipase C, but may be converted into soluble DBH by proteolysis in a stepwise manner; Da4II----Da4I----Dna4. Incubation of soluble DBH with various phospholipids did not produce any amphiphilic form. Several bands corresponding to the catalytic subunits of bovine DBH were observed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but this multiplicity was not simply correlated with the amphiphilic character of the enzyme. In the case of human DBH, we observed two bands of 78 and 84 kDa. As previously reported by others, the presence of the heavy subunit characterizes the amphiphilic forms of the enzyme. We discuss the nature of the hydrophobic domain, which could be an uncleaved signal peptide, and the organization of the different amphiphilic and nonamphiphilic DBH forms. We present two models in which dimers may possess either one hydrophobic domain or two domains belonging to each subunit; in both cases, a single detergent micelle would be bound per dimer.

Regulation of phospho enolpyruvate carboxylase from Crassula argentea: Effect of incubation with ligands and dilution on oligomeric state, activity, and allosteric properties

The relationship between the aggregation state and allosteric properties of purified phospho enolpyruvate carboxylase from Crassula argentea was examined using both kinetic and physical techniques. Analysis by native polyacrylamide gel electrophoresis showed that dilution induced a dissociation of the active tetramer to a less active dimer. Kinetic assays showed that inhibition of phospho enolpyruvate carboxylase by 5 m m malate measured at a saturating phospho enolpyruvate concentration rose to nearly 80% with increasing preassay dilution while the activity in the absence of malate remained constant. Kinetic bursts were observed when enzyme-initiated assays were measured at a subsaturating phospho enolpyruvate concentration. At saturating phospho enolpyruvate concentrations, however, increasing lags developed in response to increasing the preassay dilution of the enzyme. Further, dynamic laser-light scattering measurements showed that preincubation of the dilute enzyme with phospho enolpyruvate stabilized the tetramer while the presence of malate induced dimer formation. These observations confirm and extend earlier work with the extracted active malate insensitive night and less active, malate-sensitive day forms of the enzyme (Wu and Wedding [1985] Plant Physiol. 77, 667–675). Activity measured at subsaturating phospho enolpyruvate concentrations dropped with increasing preassay dilution of enzyme, while activation by 3.2 m m glucose 6-phosphate, assayed at a low phospho enolpyruvate concentration (0.044 m m), increased with dilution to nearly 400%. In this case activation results from a decrease in the control rate as the activity measured in the presence of glucose 6-phosphate was nearly constant, similar in effect to saturating phospho enolpyruvate in the assay. Glucose 6-phosphate induced tetramer formation of the dilute enzyme as measured by light-scattering similar to the effects induced by PEP. In addition, when diluted (dimeric) PEPC was preincubated with PEP or glucose 6-phosphate the enzyme became less sensitive to malate inhibition, while the active-site directed ligand 2-phosphoglycolate had no effect on malate inhibition. These results indicate that both the substrate PEP and the activator glucose 6-phosphate stabilize the active tetramer via binding and interaction at an activator site separate from the active site.

Human platelet factor 4 subunit association/dissociation thermodynamics and kinetics

Platelet factor 4 (PF4) monomers (7800 daltons) form dimers and tetramers in varying molar ratios under certain solution conditions [Mayo, K. H., & Chen, M. J. (1989) Biochemistry 28, 9469]. The presence of a simplified aromatic region (one Tyr and two His) and resolved monomer, dimer, and tetramer Y60 3,5 ring proton resonances makes study of PF4 aggregate association/dissociation thermodynamics and kinetics possible. PF4 protein subunit association/dissociation equilibrium thermodynamic parameters have been derived by 1H NMR (500MHz) resonance line-fitting analysis of steady-state Y60 3,5 ring proton resonance monomer-dimer-tetramer populations as a function of temperature from 10 to 40 degrees C. Below 10 degrees C and above 40 degrees C, resonance broadening and overlap severely impaired analysis. Enthalpic and entropic contributions to dimer association Gibb's free energy [-5.1 kcal/mol (30 degrees C)] are +2.5 +/- 1 kcal/mol and +26 +/- 7 eu, respectively, and for tetramer association Gibb's free energy [-5.7 kcal/mol (30 degrees C)], they are -7.5 +/- 1 kcal/mol and -7 +/- 3 eu, respectively. These thermodynamic parameters are consistent with low dielectric medium electrostatic/hydrophobic interactions governing dimer formation and hydrogen bonding governing tetramer formation. Association/dissociation kinetic parameters, i.e., steady-state jump rates, have been derived from exchange-induced line-width increases and from 1H NMR (500 MHz) saturation-transfer and spin-lattice (Tl) relaxation experiments. From dissociation jump rates and equilibrium constants, association rate constants were estimated. For dimer and tetramer equilibria at 30 degrees C, unimolecular dissociation rate constants are 35 +/- 10 s-1 for dimer dissociation and 6 +/- 2 s-1 for tetramer dissociation.(ABSTRACT TRUNCATED AT 250 WORDS)

The interaction of troponin C with phosphofructokinase. Comparison with calmodulin

Phosphofructokinase (PFK) is a calmodulin (CaM)-binding protein [Mayr & Heilmeyer (1983) FEBS Lett. 195, 51-57]. We found that troponin C (TnC), which is homologous to CaM, also binds PFK and affects PFK's catalytic activity, aggregation states and conformational changes as CaM does in most cases. PFK titration of N-acetylaminoethyl-5-naphthylamido-1-sulphonate ('AEDANS')-TnC showed that its apparent dissociation constant is comparable with that of PFK-CaM. Fluorescent labels were also used to probe contact regions on TnC and CaM. It is likely that the C-terminal end of the connecting strand of the TnC molecule is close to PFK in the binary complex. Hydrophobic regions of TnC and CaM also possibly play roles in the binding and polymerization of PFK. TnC and CaM deactivate PFK through accelerating PFK conformational change as well as through accelerating PFK tetramer dissociation, as implied in the results of activity, light-scattering, fluorescence and c.d. experiments. The intact molecule of CaM appears to be required to deactivate PFK, because neither half of the CaM molecule has an effect on PFK activity.

Monomer-tetramer equilibrium of the Escherichia coli ssb-1 mutant single strand binding protein

The Escherichia coli single strand binding (SSB) protein is an essential protein required for DNA replication and involved in recombination and a number of repair processes. It is a stable homotetramer in solution; however the ssb-1 mutation (His-55 to Tyr) destabilizes the tetramer with respect to monomers and this defect seems to explain the observed phenotype (Williams, K. R., Murphy, J. B., and Chase, J. W. (1984) J. Biol. Chem. 259, 11804-11811). We report a quantitative study of the SSB-1 monomer-tetramer equilibrium in vitro as a function of temperature, pH, NaCl, MgCl2, urea, and guanidine hydrochloride concentrations. The self-assembly equilibrium was monitored by the increase in intrinsic protein fluorescence anisotropy accompanying the formation of the tetramer. The experimental isotherms indicate that SSB-1 dimers are not highly populated at equilibrium, hence the formation of the tetramer is well-described as a one-step association of four monomers. At 25 degrees C, pH 8.1, the monomer concentration for 50% tetramer dissociation is (MT)1/2 = 0.87 microM, corresponding to a monomer-tetramer equilibrium constant, KT = 3 +/- 1 x 10(18) M-3. The tetramerization constant, KT, is highly dependent upon temperature and pH, with delta H0 = -51 +/- 7 kcal/mol (pH 8.1) and delta H0 = -37 +/- 5 kcal/mol (pH 6.9). There is no effect of NaCl on the monomer-tetramer association in the range from 0.20 to 1.0 M; however, MgCl2 decreases the stability of the SSB-1 tetramer. In the presence of high concentrations of the single-stranded oligonucleotide, dT(pT)15, the tetramerization constant is slightly increased indicating that binding of the oligonucleotide to the SSB-1 monomer promotes the assembly process, although not dramatically. The large negative delta H0 that is associated with formation of the tetramer provides a likely explanation for the temperature sensitivity of the ssb-1 mutation.

Arc repressor is tetrameric when bound to operator DNA

The Arc repressor of bacteriophage P22 is a member of a family of DNA-binding proteins that use N-terminal residues in a beta-sheet conformation for operator recognition. Here, Arc is shown to bind to its operator site as a tetramer. When mixtures of Arc (53 residues) and an active variant of Arc (78 residues) are used in gel retardation experiments, five discrete protein-DNA complexes are observed. This result is as expected for operators bearing heterotetramers containing 4:0, 3:1, 2:2, 1:3, and 0:4 ratios of the two proteins. Direct measurements of binding stoichiometry support the conclusion that Arc binds to a single 21-base-pair operator site as a tetramer. The Arc-operator binding reaction is highly cooperative (Hill constant = 3.5) and involves at least two coupled equilibria. In the first reaction, two unfolded monomers interact to form a folded dimer (Bowie & Sauer, 1989a). Rapid dilution experiments indicate that the Arc dimer is the kinetically significant DNA-binding species and allow an estimate of the equilibrium dissociation constant for dimerization [K1 = 5 (+/- 3) x 10(-9) M]. The rate of association of Arc-operator complexes shows the expected second-order dependence on the concentration of free Arc dimers, with k2 = 2.8 (+/- 0.7) x 10(18) M-2 s-1. The dissociation of Arc-operator complexes is a first-order process with k-2 = 1.6 (+/- 0.6) x 10(-4) s-1. The ratio of these kinetic constants [K2 = 5.7 (+/- 2.3) x 10(-23) M2] provides an estimate for the equilibrium constant for dissociation of the DNA-bound tetramer to two free Arc dimers and the operator. An independent determination of this complex equilibrium constant [K2 = 7.8 (+/- 4.8) x 10(-23) M2] was obtained from equilibrium binding experiments.

Thermal denaturation of .beta.-galactosidase and of two site specific mutants

The thermal denaturation of wild-type beta-galactosidase and two beta-galactosidases with substitutions at the active site was studied by kinetics, differential scanning calorimetry, electrophoresis, molecular exclusion chromatography, and circular dichroism. From the results, a model is developed for thermal denaturation of beta-galactosidase which includes the reversible dissociation of ligands, reversible formation of an inactive tetramer, irreversible dissociation of the inactive tetramer to inactive monomers, and subsequent aggregation of inactive monomers to dimers and larger aggregates. Under some conditions, partial reversibility of the activity loss could be demonstrated, and several intermediates in the thermal denaturation process were trapped by quenching and observed by electrophoresis and molecular exclusion chromatography. The ligands Mg2+ and phenylethyl thio-beta-D-galactoside increase the stability of beta-galactosidase to heat denaturation by shifting the ligand binding equilibrium according to Le Chatelier's principle, thus decreasing the concentration of the ligand-free tetramer which can proceed to subsequent steps. Circular dichroism results indicated that beta-galactosidase is dominated by beta-sheet with lower amounts of alpha-helix. Large changes in secondary structure begin to occur only after activity has been lost. Single amino acid changes at the active site can have significant effects on thermal stability of beta-galactosidases. Some of the effects result from increased thermal stability of the ligand-free enzyme itself. Other effects result from changes in ligand binding, but the magnitude of the resulting changes in stability is not related to the strength of ligand binding in a simple fashion.

Double-mixing kinetic studies of the reactions of methyl isocyanide and carbon monoxide with diliganded intermediates of hemoglobin: .alpha.2CO.beta.2 and .alpha.2.beta.2CO

Kinetics of the reactions of CO and methyl isocyanide with two diliganded intermediates of hemoglobin, alpha 2CO beta 2 and alpha 2 beta 2CO, have been studied by double-mixing and microperoxidase methods. The valency hybrids were prepared by high-pressure liquid chromatography. The reaction time courses of ligand combination and dissociation with both of the ligands were biphasic, and in CO combination reaction the zero-time amplitudes of the two phases were independent of the protein concentration. In the presence of 2 M urea the reaction time course was clearly dependent on protein concentration, as the zero-time amplitude of the fast phase increased at lower protein concentrations. These two observations indicate that little dissociation of tetramers into dimers occurs in the absence of urea. Consistent with this, the kinetic data for the reactions of CO best fit a reaction model consisting of two tetrameric species not in rapid equilibrium with each other. Various considerations, however, suggest that the reaction model is more appropriately described as 2D in equilibrium R in equilibrium T. The reaction of triliganded species (Hb4(CO)2Me1) with methyl isocyanide was monophasic, and the reaction model suggested a fast T in equilibrium R structural change after the binding of the third ligand. Although the precise structural nature of the two species remains undefined, it is concluded that the biphasicity in the reactions of the two hybrids is characteristic of the diliganded species only and is independent of the nature of the ligand.

Urea-induced inactivation, dissociation and unfolding of the allosteric phosphofructokinase from Escherichia coli

The influence of urea on the allosteric phosphofructokinase from Escherichia coli has been studied by measuring the changes in enzymatic activity, protein fluorescence, circular dichroism, and retention in size-exclusion chromatography. Tetrameric, dimeric, and monomeric forms of the protein can be discriminated by their elution from a high-performance liquid chromatography gel filtration column. Three successive steps can be detected during the urea-induced denaturation of phosphofructokinase: (i) the dissociation of the native tetramer into dimers which abolishes the activity; (ii) the dissociation of dimers into monomers which exposes the unique tryptophan, Trp-311, to the aqueous solvent; (iii) the unfolding of the monomers which disrupts most of the secondary structure. This pathway involves the ordered dissociation of the interfaces between subunits and supports a previous hypothesis (Deville-Bonne et al., 1989). Phosphofructokinase can be quantitatively renatured from urea solutions, provided that precautions are taken to avoid the aggregation of one insoluble monomeric state. The renaturation of phosphofructokinase from urea implies three steps: an initial folding reaction within the monomeric state is followed by two successive association steps. The faster association step restores the native fluorescence, and the slower regenerates the active enzyme. The renaturation and denaturation of phosphofructokinase correspond to the complex pathway: tetramer in equilibrium dimer in equilibrium folded monomer in equilibrium unfolded monomer. It is found that the subunit interface which forms the regulatory site is more stable and associates 40 times more rapidly than the subunit interface which forms the active site.

Effect of long wave u.v. irradiation on the affinity of haemoglobin for oxygen

Changes in the slope of haemoglobin-oxygen dissociation curve and its position were studied before and after the influence of long wave u.v. irradiation. Haemoglobin showed a lower than normal affinity for oxygen when exposed to 5.45 × 10 −3 J/cm 2 and to lesser extent to doses of 10.90 × 10 −3 J/cm 2. The elevation in P 50 (representing PO 2 at which Hb is half saturated) at these doses is mainly due to the new acidic groups which, by unfolding of this globular protein, become exposed in its surface. The fall in P 50 at relatively high doses was found as a result of methaemoglobin increase and the partial dissociation of Hb tetramer to dimer and monomer.

Plasma retention and metabolic fate of hemoglobin modified with an interdimeric covalent cross link.

Hemoglobin (Hb) modified with an interdimeric bicovalent cross link using 2-nor-2-formylpyridoxal 5'-phosphate (NFPLP) as the cross-linking agent has an exceptionally low O2 affinity (P50 = 47 torr), enabling it to deliver more oxygen at tissue pO2 than blood. In addition, the covalent cross link prevents dissociation of the HbXL tetramers. By using 3H-labeled HbXL, the present study investigated intravascular retention time of cross linked Hb (HbXL), organ distribution, and routes by which HbXL is metabolized and eliminated from the body. The rats were injected with an i.v. bolus (125-200 mg Hb/kg body weight) of either 3H-labeled HbXL or noncross-linked pyridoxal 5'-phosphate modified Hb (diPLPHb) as a control. Urine and feces were collected daily for up to 9 days. Organs and tissues were harvested either at 9 hr or 9 days and assayed for 3H-label content by standard liquid scintillation counting. Plasma retention of HbXL at this dose was about three times longer than diPLPHb, and no HbXL as such was recovered in the urine. HbXL did undergo metabolic degradation in the body, with labeled fragments (mol. wt. less than 10,000) being excreted by the kidneys and gastrointestinal tract. Total body clearance of the label by 9 days amounted to approximately 83% of the injected dose.

The effects of p-mercuribenzenesulfonate on purified spectrin and actin

The compound p-mercuribenzenefulfonate was found to affect the self-association behavior of both spectrin and actin. The reagent brings about the depolymerization of F-actin, as judged from the decrease in the fluorescence of an attached pyrene label, with a second-order rate constant an order of magnitude less than that for the disruption of isolated erythrocyte cytoskeletons. Therefore, it is unlikely that the depolymerization of actin is the rate-determining step in the mercurial-dependent disruption of the erythrocyte cytoskeleton. Low reagent concentrations caused an initial rapid dissociation of spectrin tetramers at a rate comparable with that of cytoskeleton disruption. Prolonged incubation, or higher reagent concentrations, resulted in subsequent aggregation of spectrin. The reagent also prevented the interaction between spectrin and actin, presumably through its depolymerization of actin and its effects on spectrin. The early event in the disruption of isolated erythrocyte cytoskeletons by p-mercuribenzenesulfonate thus appears to be the dissociation of spectrin oligomers. Subsequent depolymerization of actin brought about by the reagent then results in total disruption of the cytoskeleton.