Two-dimensional infrared correlation spectroscopy study of sequential events in the heat-induced unfolding and aggregation process of myoglobin

Unfolding and aggregation are basic problems in protein science with serious biotechnological and medical implications. Probing the sequential events occurring during the unfolding and aggregation process and the relationship between unfolding and aggregation is of particular interest. In this study, two-dimensional infrared (2D IR) correlation spectroscopy was used to study the sequential events and starting temperature dependence of Myoglobin (Mb) thermal transitions. Though a two-state model could be obtained from traditional 1D IR spectra, subtle noncooperative conformational changes were observed at low temperatures. Formation of aggregation was observed at a temperature (50-58 degrees C) that protein was dominated by native structures and accompanied with unfolding of native helical structures when a traditional thermal denaturation condition was used. The time course NMR study of Mb incubated at 55 degrees C for 45 h confirmed that an irreversible aggregation process existed. Aggregation was also observed before fully unfolding of the Mb native structure when a relative high starting temperature was used. These findings demonstrated that 2D IR correlation spectroscopy is a powerful tool to study protein aggregation and the protein aggregation process observed depends on the different environmental conditions used.     

Glutamine deamidation destabilizes human gammaD-crystallin and lowers the kinetic barrier to unfolding

Human eye lens transparency requires life long stability and solubility of the crystallin proteins. Aged crystallins have high levels of covalent damage, including glutamine deamidation. Human gammaD-crystallin (HgammaD-Crys) is a two-domain beta-sheet protein of the lens nucleus. The two domains interact through interdomain side chain contacts, including Gln-54 and Gln-143, which are critical for stability and folding of the N-terminal domain of HgammaD-Crys. To test the effects of interface deamidation on stability and folding, single and double glutamine to glutamate substitutions were constructed. Equilibrium unfolding/refolding experiments of the proteins were performed in guanidine hydrochloride at pH 7.0, 37 degrees C, or urea at pH 3.0, 20 degrees C. Compared with wild type, the deamidation mutants were destabilized at pH 7.0. The proteins populated a partially unfolded intermediate that likely had a structured C-terminal domain and unstructured N-terminal domain. However, at pH 3.0, equilibrium unfolding transitions of wild type and the deamidation mutants were indistinguishable. In contrast, the double alanine mutant Q54A/Q143A was destabilized at both pH 7.0 and 3.0. Thermal stabilities of the deamidation mutants were also reduced at pH 7.0. Similarly, the deamidation mutants lowered the kinetic barrier to unfolding of the N-terminal domain. These data indicate that interface deamidation decreases the thermodynamic stability of HgammaD-Crys and lowers the kinetic barrier to unfolding due to introduction of a negative charge into the domain interface. Such effects may be significant for cataract formation by inducing protein aggregation or insolubility.     

Studies on (Na+ + K+)-activated ATPase. XXXVIII. A 100 000 molecular weight protein as the low-energy phosphorylated intermediate of the enzyme.

Phosphorylation of NaI-treated bovine brain cortex microsomes by inorganic phosphate in the presence of Mg2+ and ouabain has been studied at 0 degrees C (pH 7.4) and 20 degrees C (pH 7.0). Nearly maximal (90%) and half-maximal phosphorylation are achieved at 20 degrees C within 2 min with 50--155 and 5.6--17 muM 32Pi, respectively, and at 0 degrees C within 75 s with 300--600 and 33--66 muM 32Pi, respectively. Maximal phosphorylation yields 146 pmol 32P - mg-1 protein. Without ouabain (20 degrees C, pH 7.0) less than 25% of the incorporation observed in the presence of ouabain is reached. Preincubation of the native microsomes with Mg2+ and K+, in order to decompose possibly present high-energy phosphoryl-bonds prior to ouabain treatment, does not affect the maximal phosphate incorporation. This indicates that the inorganic phosphate incorporation is not due to an exchange with high-energy phosphoryl-bonds, which might have been preserved in the microsomal preparations. Phosphorylation of the native microsomes by ATP in the presence of Mg2+ and Na+ reaches 90 and 50% maximal levels within 15--30 s at 0 degrees C and pH 7.4 at concentrations of [gamma-32P]ATP of 5--32 and 0.5--3.5 muM, respectively. The maximal phosphorylation level is 149 pmol 32P-mg-1 protein, equal to that of ouabain-treated microsomes phosphorylated by inorganic phosphate. Both inorganic phosphate and ATP phosphorylate on site per active enzyme subunit of 135 000 molecular weight. From the equilibrium constants for the phosphorylation of ouabain-treated microsomes by inorganic phosphate at 0 degrees C and 20 degrees C standard free-energy changes of --5.4 and --6.8 kcal/mol, respectively, are calculated. These values yield a standard enthalpy change of 14 kcal/mol and an entropy change of 70 cal/mol - degree K. This characterizes the reaction as a process driven by an entropy change. The intermediate formed by phosphorylation with Pi has maximal stability at acidic pH, as is the case for the intermediate formed with ATP. Solubilization in sodium dodecyl sulfate stabilizes the phosphoryl-bond in the pH range of 4--7. The non-solubilized preparation has optimal stability at pH 2--4, the level of which is equal to that of detergent-solubilized intermediate. Sodium dodecyl sulfate gel electrophoresis of the microsomes at pH 3, following incorporation of 32Pi yields 11 protein bands, only one of which (mol. wt 100 000--106 000) carries the radioactive label. This protein has the same molecular weight as the protein, which is phosphorylated by ATP in the presence of Mg2+ and Na+.     

Salts and glycine increase reversibility and decrease aggregation during thermal unfolding of ribonuclease-A

Ribonuclease-A (RNase-A) has been a model for studying protein folding and unfolding. However, we show here that its unfolding at neutral pH is complicated by aggregation. Circular dichroism thermal scans showed that reversibility of RNase-A after heating is only about 63%. In accordance with this observation, native-polyacrylamide gel electrophoresis of the sample heated at 75 degrees C showed formation of soluble oligomers. Ammonium sulfate at 0.4 M caused about a 3 degrees C higher melting temperature and nearly complete reversibility, while glycine and NaCl at 0.4 M significantly increased reversibility and decreased aggregation without affecting melting temperature. These results demonstrate that aggregation makes thermal unfolding of RNase-A at least partially irreversible and salts and glycine increase reversibility and decrease aggregation.     

Examination of the structure, stability, and catalytic potential in the engineered phosphoryl carrier domain of pyruvate phosphate dikinase

Pyruvate phosphate dikinase (PPDK) is a multidomain protein that catalyzes the interconversion of ATP, pyruvate, and phosphate with AMP, phosphoenolpyruvate (PEP), and pyrophosphate using its central domain to transport phosphoryl groups between two distant active sites. In this study, the mechanism by which the central domain moves between the two catalytic sites located on the N-terminal and C-terminal domains was probed by expressing this domain as an independent protein and measuring its structure, stability, and ability to catalyze the ATP/phosphate partial reaction in conjunction with the engineered N-terminal domain protein (residues 1-340 of the native PPDK). The encoding gene was engineered to express the central domain as residues 381-512 of the native PPDK. The central domain was purified and shown to be soluble, monomeric (13,438 Da), and stable (deltaG = 4.3 kcal/mol for unfolding in buffer at pH 7.0, 25 degrees C) and to possess native structure, as determined by multidimensional heteronuclear NMR analysis. The main chain structure of the central domain in solution aligns closely with that of the X-ray structure of native PPDK (the root-mean-square deviation is 2.2 A). Single turnover reactions of [14C]ATP and phosphate, carried out in the presence of equal concentrations of central domain and the N-terminal domain protein, did not produce the expected products, in contrast to efficient product formation observed for the N-terminal central domain construct (residues 1-553 of the native PPDK). These results are interpreted as evidence that the central domain, although solvent-compatible, must be tethered by the flexible linkers to the N-terminal domain for the productive domain-domain docking required for efficient catalysis.     

pH dependence of the reversible and irreversible thermal denaturation of gamma interferons

Heated at pH 6.0 and at 50 degrees C, human interferon gamma (HuIFN-gamma) is inactivated via the formation of insoluble aggregates. At pH 6.0, the aggregation rate increases with temperature from 40 to 65 degrees C. There is a temperature-dependent time lag to aggregate formation observed in the generation of light-scattering particles at pH 6.0, and this correlates with the fast phase observed in the kinetics of reversible thermal unfolding. In addition, the dependence of aggregation kinetics on temperature closely follows the reversible melting curve. These observations suggest that at pH 6.0 irreversible thermal denaturation and aggregation depend on partial or complete unfolding of the molecule. At pH 5.0, also at 50 degrees C, the molecule is stable to irreversible aggregation. In reversible unfolding in 0.25 M guanidine hydrochloride, the Tm for HuIFN-gamma increases from 30.5 degrees C at pH 4.75 to 41.8 degrees C at pH 6.25, in analogy to the behavior of other globular proteins. These observations suggest that the relative instability of HuIFN-gamma to irreversible denaturation via aggregation at pH 6.0 compared to pH 5.0 is not due to an increased stability toward unfolding at the lower pH. Alternatively, stability at pH 5.0 must be due either to the improved solution properties of the unfolded state or to the improved solubility/decreased kinetic lifetime of an unfolding intermediate. Aggregation of HuIFN-gamma at 50 degrees C is half-maximal at pH 5.7, suggesting that protonation of one or both of the histidine residues may be involved in this stabilization.(ABSTRACT TRUNCATED AT 250 WORDS)     

Denaturation of thymidylate synthetase from amethopterin-resistant Lactobacillus casei.

The effects of various concentrations of urea and guanidine hydrochloride on enzyme activity and on subunit association were determined. Incubation of thymidylate synthetase with buffered solutions of 3M to 3.5M guanidine hydrochloride or 5 M to 6 M urea resulted in the loss of about 90% of the enzyme activity. Under these denaturing conditions a red shift of the fluorescence emission maximum from 340 nm to 351 nm was observed together with a significant decrease in the relative fluorescence intensity of the protein. Studies at both 4 degrees C and 25 degrees C indicated that the enzyme was in the dimer form in 2 M guanidine hydrochloride but was dissociated into monomers in concentrations of this denaturant of 3 M and above. Although only monomeric species were evident at 4 degrees C in 6 M urea, at 25 25 degrees C this denaturant caused protein aggregation which increased with decreasing phosphate buffer concentration. Enzyme (5 mg/ml) in 0.5 M potassium phosphate buffer, pH 6.8, containing 4 M guanidine hydrochloride gave a minimum S20, w value of 1.22S at 25 degrees C. Sedimentation behavior of the native enzyme in the range of 5 to 20 mg/ml was only slightly concentration-dependent (4.28 S to 4.86 S) but extensive aggregation occurred above 20 mg/ml.     

Aggregation of granulocyte colony stimulating factor under physiological conditions: characterization and thermodynamic inhibition

We have investigated the aggregation of recombinant human granulocyte colony stimulating factor (rhGCSF), a protein that rapidly aggregates and precipitates at pH 6.9 and 37 degrees C. We observed that native monomeric rhGCSF reversibly forms a dimer under physiological conditions and that this dimeric species does not participate in the irreversible aggregation process. Sucrose, a thermodynamic stabilizer, inhibits the aggregation of rhGCSF. We postulate that sucrose acts by reducing the concentration of structurally expanded species, consistent with the hypothesis that preferential exclusion favors most compact species in the native state ensemble. Thermodynamic stability data from unfolding curves and hydrogen-deuterium exchange experimental results support the above hypothesis. Thus, the strategy of stabilizing the native state of the protein under physiological conditions using thermodynamic stabilizers, especially ligands binding with high affinity to the native state, is expected to protect against protein aggregation occurring under such nonperturbing solution conditions.     

Thermodynamic characterization of an intermediate state of human growth hormone.

The thermal denaturation of recombinant human growth hormone (rhGH) was studied by differential scanning calorimetry and circular dichroism spectroscopy (CD). The thermal unfolding is reversible only below pH 3.5, and under these conditions a single two-state transition was observed between 0 and 100 degrees C. The magnitudes of the deltaH and deltaCp of this transition indicate that it corresponds to a partial unfolding of rhGH. This is also supported by CD data, which show that significant secondary structure remains after the unfolding. Above pH 3.5 the thermal denaturation is irreversible due to the aggregation of rhGH upon unfolding. This aggregation is prevented in aqueous solutions of alcohols such as n-propanol, 2-propanol, or 1,2-propanediol (propylene glycol), which suggests that the self-association of rhGH is caused by hydrophobic interactions. In addition, it was found that the native state of rhGH is stable in relatively high concentrations of propylene glycol (up to 45% v/v at pH 7-8 or 30% at pH 3) and that under these conditions the thermal unfolding is cooperative and corresponds to a transition from the native state to a partially folded state, as observed at acidic pH in the absence of alcohols. In higher concentrations of propylene glycol, the tertiary structure of rhGH is disrupted and the cooperativity of the unfolding decreases. Moreover, the CD and DSC data indicate that a partially folded intermediate with essentially native secondary structure and disordered tertiary structure becomes significantly populated in 70-80% propylene glycol.     

The heat denaturation of bacterial ribonuclease studied by infrared spectroscopy]

The thermal denaturation of bacterial ribonuclease in the interval of pH 2.5-7.0 has been investigated by means of infra-red spectroscopy method. The protein melting for pH 2.5 begins at the temperature 25 degrees C and is accompanied by secondary protein structure reconstruction, partially destroying native beta-structure and leading to new denatured conformation appearance of different types of beta-turns. Spectral changes for pH 3.5 and 7.0 are significantly less in the same frequency areas. At the temperature more than 50 degrees C protein aggregation takes place with inter-molecule-beta-form formation.     

A kinetic study of the folding of staphylococcal nuclease using size-exclusion chromatography.

The kinetics of the hydrodynamic volume change accompanying the reversible unfolding of staphylococcal nuclease have been observed by size-exclusion chromatography at 4 degrees C and pH 7.0 using the denaturant guanidine hydrochloride. The observed chromatographic profiles have been simulated by a six-component unfolding/refolding mechanism using a consistent set of equilibrium and kinetic parameters. The native protein is an equilibrium mixture of the cis and trans isomers of the peptide bond preceding proline-117. The native conformation containing the cis isomer dominates the equilibrium mixture, is more stable, and unfolds more slowly at its transition midpoint. The denatured protein is an equilibrium mixture of at least four components, the cis/trans isomers of proline-117 and one of the five remaining prolines. The dominant refolding pathway is initiated from the denatured component containing the trans isomer of proline-117. The six-component mechanism is consistent with tryptophan fluorescence kinetic measurements of the wild-type protein and with chromatographic measurements of a mutant P117G protein.     

Protein thermal aggregation involves distinct regions: sequential events in the heat-induced unfolding and aggregation of hemoglobin

Protein thermal aggregation plays a crucial role in protein science and engineering. Despite its biological importance, little is known about the mechanism and pathway(s) involved in the formation of aggregates. In this report, the sequential events occurring during thermal unfolding and aggregation process of hemoglobin were studied by two-dimensional infrared correlation spectroscopy. Analysis of the infrared spectra recorded at different temperatures suggested that hemoglobin denatured by a two-stage thermal transition. At the initial structural perturbation stage (30-44 degrees C), the fast red shift of the band from alpha-helix indicated that the native helical structures became more and more solvent-exposed as temperature increased. At the thermal unfolding stage (44-54 degrees C), the unfolding of solvent-exposed helical structures dominated the transition and was supposed to be responsible to the start of aggregation. At the thermal aggregation stage (54-70 degrees C), the transition was dominated by the formation of aggregates and the further unfolding of the buried structures. A close inspection of the sequential events occurring at different stages suggested that protein thermal aggregation involves distinct regions.     

The role of epsilon-amino groups of lysine of human serum albumin in heat-induced protein denaturation. Increased stability of glycosylated and alkylated albumin in aggregation during heating

Human serum albumin was glycosidated by prolonged protein incubation in phosphate buffer, pH 6.8-7.0, with excess glucose at 37 degrees C. epsilon-amino groups of lysine residues of the albumin molecule were alkylated by pyridoxal-5-phosphate in the presence of NaBH4. The solutions of glycosidated and alkylated serum albumin were incubated at different temperature values in the range of 20 to 80 degrees C in phosphate buffer, pH 7.0, over 30 min. The nondenatured monomer and the resulting aggregated were isolated by TSK-HW-55-gel column chromatography and polyacrylamide gel electrophoresis. The stability of modified proteins elevated in parallel to the increase in the number of the ligand molecules covalently bound to albumin amino groups. The 1-3% aqueous solutions of glycosidated serum albumin containing 3-4 glucose residues and those of alkylated albumin containing 6-7 residues of pyridoxal-5-phosphate were stable on heating up to 80 degrees C and did not form aggregates. Under these conditions the initial serum albumin completely aggregated. Preincubation of the aggregated albumin with glucose at 37 degrees C resulted in protein "renaturation" to the monomeric form with a small number of dimers and trimers.     

The unfolding enthalpy of the pH 4 molten globule of apomyoglobin measured by isothermal titration calorimetry

The unfolding enthalpy of the pH 4 molten globule from sperm whale apomyoglobin has been measured by isothermal titration calorimetry, using titration to acid pH. The unfolding enthalpy is close to zero at 20 degrees C, in contrast both to the positive values expected for peptide helices and the negative values reported for holomyoglobin and native apomyoglobin. At 20 degrees C, the hydrophobic interaction should make only a small contribution to the unfolding enthalpy according to the liquid hydrocarbon model. Our result indicates that some factor present in the unfolding enthalpies of native proteins makes the unfolding enthalpy of the pH 4 molten globule less positive than expected from data for peptide helices.     

Redox properties of the couple compound I/native enzyme of myeloperoxidase and eosinophil peroxidase.

The standard reduction potential of the redox couple compound I/native enzyme has been determined for human myeloperoxidase (MPO) and eosinophil peroxidase (EPO) at pH 7.0 and 25 degrees C. This was achieved by rapid mixing of peroxidases with either hydrogen peroxide or hypochlorous acid and measuring spectrophotometrically concentrations of the reacting species and products at equilibrium. By using hydrogen peroxide, the standard reduction potential at pH 7.0 and 25 degrees C was 1.16 +/- 0.01 V for MPO and 1.10 +/- 0.01 V for EPO, independently of the concentration of hydrogen peroxide and peroxidases. In the case of hypochlorous acid, standard reduction potentials were dependent on the hypochlorous acid concentration used. They ranged from 1.16 V at low hypochlorous acid to 1.09 V at higher hypochlorous acid for MPO and from 1.10 V to 1.03 V for EPO. Thus, consistent results for the standard reduction potentials of redox couple compound I/native enzyme of both peroxidases were obtained with all hydrogen peroxide and at low hypochlorous acid concentrations: possible reasons for the deviation at higher concentrations of hypochlorous acid are discussed. They include instability of hypochlorous acid, reactions of hypochlorous acid with different amino-acid side chains in peroxidases as well as the appearance of a compound I-chloride complex.     

Thermodynamic effects of disulfide bond on thermal unfolding of the starch-binding domain of Aspergillus niger glucoamylase

The thermodynamic effects of the disulfide bond of the fragment protein of the starch-binding domain of Aspergillus niger glucoamylase was investigated by measuring the thermal unfolding of the wild-type protein and its two mutant forms, Cys3Gly/Cys98Gly and Cys3Ser/Cys98Ser. The circular dichroism spectra and the thermodynamic parameters of binding with beta-cyclodextrin at 25 degrees C suggested that the native structures of the three proteins are essentially the same. Differential scanning calorimetry of the thermal unfolding of the proteins showed that the unfolding temperature t1/2 of the two mutant proteins decreased by about 10 degrees C as compared to the wild-type protein at pH 7.0. At t1/2 of the wild-type protein (52.7 degrees C), the mutant proteins destabilized by about 10 kJ mol(-1) in terms of the Gibbs energy change. It was found that the mutant proteins were quite stabilized in terms of enthalpy, but that a higher entropy change overwhelmed the enthalpic effect, resulting in destabilization.     

Acidic conditions stabilise intermediates populated during the folding of Im7 and Im9

The helical bacterial immunity proteins Im7 and Im9 have been shown to fold via kinetic mechanisms of differing complexity, despite having 60 % sequence identity. At pH 7.0 and 10 degrees C, Im7 folds in a three-state mechanism involving an on-pathway intermediate, while Im9 folds in an apparent two-state transition. In order to examine the folding mechanisms of these proteins in more detail, the folding kinetics of both Im7 and Im9 (at 10 degrees C in 0.4 M sodium sulphate) have been examined as a function of pH. Kinetic modelling of the folding and unfolding data for Im7 between pH 5.0 and 8.0 shows that the on-pathway intermediate is stabilised by more acidic conditions, whilst the native state is destabilised. The opposing effect of pH on the stability of these states results in a significant population of the intermediate at equilibrium at pH 6.0 and below. At pH 7.0, the folding and unfolding kinetics for Im9 can be fitted adequately by a two-state model, in accord with previous results. However, under acidic conditions there is a clear change of slope in the plot of the logarithm of the folding rate constant versus denaturant concentration, consistent with the population of one or more intermediate(s) early during folding. The kinetic data for Im9 at these pH values can be fitted to a three-state model, where the intermediate ensemble is stabilised and the native state destabilised as the pH is reduced, rationalising previous results that showed that an intermediate is not observed experimentally at pH 7.0. The data suggest that intermediate formation is a general step in immunity protein folding and demonstrate that it is necessary to explore a wide range of refolding conditions in order to show that intermediates do not form in the folding of other small, single-domain proteins.     

Flexibility and viscometric studies of globular proteins ovalbumin and ovotransferrin

The ultrasonic velocity, density and viscosity of two egg proteins, ovalbumin and ovotransferrin in phosphate buffer have been studied at the physiological pH values. The thermodynamic functions for unfolding, ellipticity, surface amino acid residues and compressibility have been obtained for thermal and chemical denaturation in these food proteins. The computed values of Huggin's constant and shape factor, at a fixed ionic strength 0.16 M are found to be in agreement with the reported values for globular proteins. The slow increase in free-energy of unfolding with temperature at a fixed pH 7 suggests uncoiling and in turn, disappearance of biological activity. It has been observed that the effects of temperature and chemical denaturant on the native protein may give rise to different conformational states. In the presence of urea and sodium dodecyl sulphate (SDS), the proteins gave the excessively denatured states at 25 degrees C and pH 7, in comparison to the thermal denatured state. The positive values of partial adiabatic compressibility (see symbol in text) beta s over the temperature range 45-75 degrees C suggest the possibility of large internal flexibility in ovotransferrin than in ovalbumin.     

Stability and folding of the ferredoxin from the hyperthermophilic archaeon Acidianus ambivalens

The ferredoxin from the thermophilic archaeon Acidianus ambivalens is a small monomeric protein containing two iron-sulfur centres, one [3Fe-4S](1+/0) and one [4Fe-4S](2+/1+). It is an intrinsically hyperstable protein, being expressed at the organism's extreme optimal growth temperature: 80 degrees C. Using spectroscopic methods we have investigated the unfolding reaction of the Acidianus ambivalens ferredoxin. No unfolding of the oxidised ferredoxin was observed at pH 7.0, even in the presence of 8 M GuHCl. Upon increasing the pH to 10.0, the unfolding transition showed a midpoint at 6.3 M GuHCl and an unfolding-free energy of 70 kJ mol(-1) in buffer (pH 10) was estimated. Kinetic-unfolding experiments showed that the polypeptide unfolding correlated with rearrangement of the iron-sulfur centres to new ones which had strong absorption maxima at 520 and 610 nm. These new, possibly linear three-iron, clusters were coordinated to the unfolded protein but degraded slowly. From thermal experiments in the presence of GuHCl we estimated the melting temperature for the Acidianus ambivalens ferredoxin in buffer (at pH 7) to be 122 degrees C. Possible structural properties that contribute to the large thermal stability of the Acidianus ambivalens ferredoxin are discussed using a three-dimensional protein model.     

An equilibrium binding study of the interaction of fructose 6-phosphate and fructose 1,6-bisphosphate with rabbit muscle phosphofructokinase.

Equilibrium binding studies of the interaction of rabbit muscle phosphofructokinase with fructose 6-phosphate and fructose 1,6-bisphosphate have been carried out at 5 degrees in the presence of 1-10 mM potassium phosphate (pH 7.0 and 8.0), 5 mM citrate (pH 7.0), or 0.22 mm adenylyl imidodiphosphate (pH 7.0 and 8.0). The binding isotherms for both fructose 6-phosphate and fructose 1,6-bisphosphate exhibit negative cooperativity at pH 7.0 and 8.0 in the presence of 1-10 mM potassium phosphate at protein concentrations where the enzyme exists as a mixture of dimers and tetramers (pH 7.0) or as tetramers (pH 8.0) and at pH 7.0 in the presence of 5 mM citrate where the enzyme exists primarily as dimers. The enzyme binds 1 mol of either fructose phosphate/mol of enzyme monomer (molecular weight 80,000). When enzyme aggregation states smaller than the tetramer are present, the saturation of the enzyme with either ligand is paralleled by polymerization of the enzyme to tetramer, by an increase in enzymatic activity and by a quenching of the protein fluorescence. At protein concentrations where aggregates higher than the tetramer predominate, the fructose 1,6-bisphosphate binding isotherms are hyperbolic. These results can be quantitatively analyzed in terms of a model in which the dimer is associated with extreme negative cooperativity in binding the ligands, the tetramer is associated with less negative cooperativity, and aggregates larger than the tetramer are associated with little or no cooperativity in the binding process. Phosphate is a competitive inhibitor of the fructose phosphate sites at both pH 7.0 and 8.0, while citrate inhibits binding in a complex, noncompetitive manner. In the presence of the ATP analog adenylyl imidodiphosphate, the enzyme-fructose 6-phosphate binding isotherm is sigmoidal at pH 7.0, but hyperbolic at pH 8.0. The characteristic sigmoidal initial velocity-fructose 6-phosphate isotherms for phosphofructokinase at pH 7.0, therefore, are due to an heterotropic interaction between ATP and fructose 6-phosphate binding sites which alters the homotropic interactions between fructose 6-phosphate binding sites. Thus the homotropic interactions between fructose 6-phosphate binding sites can give rise to positive, negative, or no cooperativity depending upon the pH, the aggregation state of the protein, and the metabolic effectors present. The available data suggest the regulation of phosphofructokinase involves a complex interplay between protein polymerization and homotropic and heterotropic interactions between ligand binding sites.