Acceleration of the NAD cyanide adduct reaction by lactate dehydrogenase: the equilibrium binding effect as a measure of the activation of bound NAD

The binary complex of NAD and lactate dehydrogenase reacts reversibly with cyanide to produce a complex (E X NAD-CN) whose noncovalent interactions are similar to those in the E X NADH complex (where E is one-fourth of the tetrameric dehydrogenase). The reaction apparently is a simple bimolecular nucleophilic addition at the 4 position of the bound nicotinamide ring; viz., cyanide does not bind to the enzyme prior to reaction. The value of the dissociation constant for E X NAD-CN is about 1 X 10(-6) M and is independent of pH over the range of 6-8. The equilibrium constant for the reaction of cyanide with E X NAD is about 400-fold larger than that for the nonenzymic process after a statistical correction. This increment in Ke is accounted for by a 220-fold increase in the rate of the forward enzymic reaction (20 M-1 s-1) as compared with an approximately 2-fold decrease for the reverse process (9 X 10(-5) s-1). Thus, the increased value of the rate constant for bond formation in the enzymic reaction is attributed to an equilibrium binding effect that is translated almost entirely into a rate effect on that step (bond formation). Since the nonenzymic reaction is sensitive to solvent composition, this equilibrium binding effect likely is produced by environmental effects at the nicotinamide/dehydronicotinamide part of the coenzyme binding site on the enzyme.     

Kinetic scheme for intermolecular RNA cleavage by a ribozyme derived from hepatitis delta virus RNA

A minimal kinetic mechanism for a trans-acting ribozyme derived from the HDV antigenomic RNA self-cleaving element was established from steady-state, pre-steady-state, single-turnover, and binding kinetics. Rate constants for individual steps, including substrate binding and dissociation, cleavage, and product release and binding, were measured at 37 degrees C at pH 8.0 in 10 mM Mg(2+) using oligonucleotides as either substrates, noncleavable analogues or 3' product mimics. A substrate containing a normal 3',5'-linkage was cleaved with a first-order rate constant (k(2)) of 0.91 min(-)(1). The association rate constant for the substrate to the ribozyme (2.1 x 10(7) M(-)(1) min(-)(1)) was at the lower range of the expected value for RNA duplex formation, and the substrate dissociated with a rate constant (1.4 min(-)(1)) slightly faster than that for cleavage. Thus the binary complex was not at equilibrium with free enzyme and substrate prior to the cleavage step. Following cleavage, product release was kinetically ordered in that the 5' product was released rapidly (>12 min(-)(1)) relative to the 3' product (6.0 x 10(-)(3) min(-)(1)). Rapid 5' product release and lack of a demonstrable binding site for the 5' product could contribute to the difficulty in establishing the ribozyme-catalyzed reverse reaction (ligation). Slow release of the 3' product was consistent with the extremely low turnover under steady-state conditions as 3' product dissociation was rate-limiting. The equilibrium dissociation constant for the substrate was 24-fold higher than that of the 3' cleavage product. A substrate with a 2',5'-linkage at the cleavage site was cleaved with a rate constant (k(2)) of 1.1 x 10(-)(2) min(-)(1). Thus, whereas cleavage of a 3',5'-linkage followed a Briggs-Haldane mechanism, 2', 5' cleavage followed a Michaelis-Menten mechanism.     

RNA binding specificity of hnRNP A1: significance of hnRNP A1 high-affinity binding sites in pre-mRNA splicing.

Pre-mRNA is processed as a large complex of pre-mRNA, snRNPs and pre-mRNA binding proteins (hnRNP proteins). The significance of hnRNP proteins in mRNA biogenesis is likely to be reflected in their RNA binding properties. We have determined the RNA binding specificity of hnRNP A1 and of each of its two RNA binding domains (RBDs), by selection/amplification from pools of random sequence RNA. Unique RNA molecules were selected by hnRNP A1 and each individual RBD, suggesting that the RNA binding specificity of hnRNP A1 is the result of both RBDs acting as a single RNA binding composite. Interestingly, the consensus high-affinity hnRNP A1 binding site, UAGGGA/U, resembles the consensus sequences of vertebrate 5' and 3' splice sites. The highest affinity 'winner' sequence for hnRNP A1 contained a duplication of this sequence separated by two nucleotides, and was bound by hnRNP A1 with an apparent dissociation constant of 1 x 10(-9) M. hnRNP A1 also bound other RNA sequences, including pre-mRNA splice sites and an intron-derived sequence, but with reduced affinities, demonstrating that hnRNP A1 binds different RNA sequences with a > 100-fold range of affinities. These experiments demonstrate that hnRNP A1 is a sequence-specific RNA binding protein. UV light-induced protein-RNA crosslinking in nuclear extracts demonstrated that an oligoribonucleotide containing the A1 winner sequence can be used as a specific affinity reagent for hnRNP A1 and an unidentified 50 kDa protein. We also show that this oligoribonucleotide, as well as two others containing 5' and 3' pre-mRNA splice sites, are potent inhibitors of in vitro pre-mRNA splicing.     

Binding of [3H]Serotonin to Skeletal Muscle Actin

We previously observed that the neurotransmitter 5-hydroxytryptamine (5-HT, serotonin) binds with high- and low-affinity interactions to an actin-like protein prepared from rat brain synaptosomes. In this study, we examined its binding to highly purified actin obtained from rabbit skeletal muscle. Monomeric G-actin bound serotonin with high and low affinities, exhibiting equilibrium dissociation constants (KD values) of 5 X 10(-5) M and 4 X 10(-3) M, respectively. The serotonin binding site on actin was distinct from those sites previously characterized for divalent cations, nucleotides, and cytochalasin alkaloids. The binding of serotonin (1 microM) to G-actin was increased as much as 26-fold by divalent cations. Potassium iodine (KI) increased the affinity of G-actin for serotonin, KD values for this binding being 3 X 10(-7) M and X 10(-5) M. Serotonin bound with even higher affinity to polymerized F-actin, with KD values of 2 X 10(-8) M and 2 X 10(-5) M. However, the total number of binding sites on F-actin was only about 4% of the number of G-actin. The binding of serotonin (0.1 microM) to G-actin could be inhibited by phenothiazines (1 microM) or reserpine (10 microM), but not by classical antagonists of serotonin receptors or by drugs that release serotonin or inhibit its uptake. The binding of serotonin to actin in vivo may participate in a contractile process related to neurotransmitter release.     

Occurrence and properties of a prostaglandin F2alpha receptor in bovine corpora lutea.

Prostaglandin F2alpha was specifically bound by a particulate fraction from bovine corpora lutea. The rate constants for the association (7.5 X 10(3) M-1 S-1) and dissociation (2.1 X 10-4 S-1) reactions gave a dissociation constant of 2.8 X 10(-8) M which is similar to that determined from a Scatchard plot of binding data at equilibrium (5 X 10(-8) M). The receptor was stable for several hours at 23 degrees C but was rapidly destroyed at 37 degrees C. The pH optimum for the binding reaction was 6.3. The receptor had high specificity for prostaglandin F2alpha and had much lower affinities for other prostaglandins. Luteinizing and follicle-stimulating hormones had no effect on the prostaglandin F2alpha-receptor interaction.     

Probing the relationship between alpha-helix formation and calcium affinity in troponin C: 1H NMR studies of calcium binding to synthetic and variant site III helix-loop-helix peptides

Three 34-residue peptides corresponding to the high-affinity calcium-binding site III and two variant sequences from the muscle protein troponin C (TnC) were synthesized by solid-phase techniques. The two variant 34-residue peptides had amino acid modifications at either the coordinating positions or both the coordinating and noncoordinating positions, which corresponded to the residues found in the low-affinity calcium-binding site II of TnC. High-field 1H NMR spectroscopy was used to monitor calcium binding to each peptide to determine the effect these amino acid substitutions had on calcium affinity. The dissociation constant of the native site III peptide (SCIII) was 3 x 10(-6) M, smaller than that of the peptide incorporating the ligands from site II (LIIL), 8 x 10(-6) M, and that with the entire site II loop (LII), 3 x 10(-3) M, which bound calcium very weakly. These calcium dissociation constants demonstrate that very minor amino acid substitutions have a significant effect on the dissociation constant and give some insight into why the dissociation constants for site III and IV in TnC are 100-fold smaller than those for sites I and II. The results suggest that the differences in coordinating ligands between sites II and III have very little effect on Ca2+ affinity and that the noncoordinating residues in the site II loop are responsible for the low affinity of site II compared to the high affinity of site III in TnC.     

Plasma protein binding interaction of prednisone and prednisolone

The plasma protein binding interaction of prednisone and prednisolone were characterized by equilibrium dialysis. Prednisone and prednisolone are bound equally but weakly to human albumin (affinity constant, K approximately equal to 1 X 10(3) M-1). Transcortin affinity for prednisolone is 10-fold greater (51.3 X 10(6) M-1) than that for prednisone (4.3 X 10(6) M-1). In competition under pharmacologic conditions, prednisolone inhibits prednisone binding to transcortin producing linear binding averaging 55%. Prednisone does not affect prednisolone binding and does not complicate pharmacokinetic studies of the latter.     

Kinetic analysis of oligodeoxyribonucleotide-directed triple-helix formation on DNA

Pyrimidine oligonucleotides recognize extended purine sequences in the major groove of double-helical DNA by triple-helix formation. The resulting local triple helices are relatively stable and can block DNA recognition by sequence-specific DNA binding proteins such as restriction endonucleases. Association and dissociation kinetics for the oligodeoxyribonucleotide 5'-CTCTTTCCTCTCTTTTTCCCC (bold C's indicate 5-methylcytosine residues) are now measured with a restriction endonuclease protection assay. When oligonucleotides are present in greater than 10-fold excess over the DNA target site, the binding reaction kinetics are pseudo first order in oligonucleotide concentration. Under our standard conditions (37 degrees C, 25 mM Tris-acetate, pH 6.8, 70 mM sodium chloride, 20 mM magnesium chloride, 0.4 mM spermine tetrahydrochloride, 10 mM beta-mercaptoethanol, 0.1 mg/mL bovine serum albumin) the value of the observed pseudo-first-order association rate constant, k2obs, is 1.8 x 10(3) +/- 1.9 x 10(2) L.(mol of oligomer-1.s-1. Measurement of the dissociation rate constant yields an equilibrium dissociation constant of approximately 10 nM. Increasing sodium ion concentration slightly decreased the association rate, substantially increased the dissociation rate, and thereby reduced the equilibrium binding constant. This effect was reversible by increasing multivalent cation concentration, confirming the significant role of multivalent cations in oligonucleotide-directed triple-helix formation under these conditions. Finally, a small reduction in association rate, a large increase in dissociation rate, and a resulting reduction in the equilibrium binding constant were observed upon increasing the pH between 6.8 and 7.2.     

Determination of free and bound microtubular protein and guanine nucleotide under equilibrium conditions.

The dissociation constant for GDP binding to the E site of tubulin isolated by chromatography on Sepharose 6B is equal to 6.1 X 10(-8) M, as determined by the Hummel-Dryer procedure. This is smaller than any previously reported value, and the discrepancy with earlir results is analyzed.By use of a recently described column centrifugation procedure [Penefsky, H. S. (1977) J. Biol. Chem. 252, 2891-2899], it was established that GDP and GTP bind to the same site. GTP is bound 2.8-fold tighter than GDP, and the dissociation constant is 2.2 X 10(-8) M. A new method for the determination of dissociation constants for a protein-bound ligand, based on a quantitative analysis of the loss of ligand during exclusion chromatography, is presented. This has been used to determine that the dissociation constant for GDP bound to tubulin is equal to 5.5 X 10(-8) M, in excellent agreement with that determined independently from the Hummel-Dryer method. A previous theoretical treatment [Dixon, H. B. F. (1976) Biochem. J. 159, 161-162] of ligand loss during exclusion chromatography is discussed.     

Binding of substrates and other anions to yeast phosphoglycerate kinase.

1. The binding of all four substrates to yeast phosphoglycerate kinase has been studied using a gel filtration technique. The binding of phosphate and sulphate anions has also been investigated. 2. Two sites for each adenine nucleotide were found, one site being weaker than the other by between 30 and 50-fold. Only one binding site for the phosphoglycerate substrates was found. 3. 1,3-Bisphosphoglycerate (1,3-P2-glycerate) bound to the enzyme approximately 1000 times tighter than the other three substrates, its dissociation constant being 0.06 micrometer at ionic strength 0.15 M. 4. Sulphate and phosphate were mutually competitive and sulphate competed with the binding of all substrates except MgADP. MgADP bound to the enzyme more weakly in the presence of sulphate. The dissociation constant for sulphate binding was 1.6 mM at ionic strength of 0.15 M, and 0.05 mM at ionic strength 0.015 M. 5. These results are consistent with sulphate acting as a competitive inhibitor, as found by kinetic studies at high sulphate concentrations. The activatory effect of sulphate at lower concentrations and the substrate activation phenomea displayed by this enzyme, are interpreted in terms of a two-step dissociation of 1, 3-P2-glycerate. The presence of moderate concentrations of MgATP, 3-phosphoglycerate or sulphate causes acceleration of the rate of dissociation of the product, 1, 3-P2-glycerate, this being the rate-limiting step in the overall enzyme reaction.     

A specific cyclic guanosine 3':5'-monophosphate-binding protein in Caulobacter crescentus.

A binding protein specific for cyclic guanosine 3':5'-monophosphate (cyclic GMP) has been partially purified from extracts of the eubacterium Caulobacter crescentus and resolved from cyclic adenosine 3':5'-monophosphate (cyclic AMP)-binding activity. Binding of cyclic GMP is not affected by the addition of cyclic AMP or 5'-GMP, but is inhibited about 50 percent by a 50-fold molar excess of dibutyryl cyclic GMP or cyclic hypoxanthine 3':5'-monophosphate. The apparent dissociation constant for the cyclic GMP-binding protein complex is 1.1 X 10(-6) M.     

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.     

Contacts between the factor TUF and RPG sequences

The yeast TUF factor binds specifically to RPG-like sequences involved in multiple functions at enhancers, silencers, and telomeres. We have characterized the interaction of TUF with its optimal binding sequence, rpg-1 (1-ACACCCATACATTT-14), using a gel DNA-binding assay in combination with methylation protection and mutagenesis experiments. As many as 10 base pairs appear to be engaged in factor binding. Analysis of a collection of 30 different RPG mutants demonstrated the importance of 8 base pairs at position 2, 3, 4, 5, 6, 7, 10, and 12 and the critical role of the central GC pair at position 5. Methylation protection data on four different natural sites confirmed a close contact at positions 4, 5, 6, and 10 and suggested additional contacts at base pairs 8, 12, and 13. The derived consensus sequence was RCAAYCCRYNCAYY. A quantitative band shift analysis was used to determine the equilibrium dissociation constant for the complex of TUF and its optimal binding site rpg-1. The specific dissociation constant (K8) was found to be 1.3 x 10(-11) M. The comparison of the K8 value with the dissociation constant obtained for nonspecific DNA sites (Kn8 = 8.7 x 10(-6) M) shows the high binding selectivity of TUF for its specific RPG target.     

Binding kinetics of ecotropic (Moloney) murine leukemia retrovirus with NIH 3T3 cells.

A quantitative analysis of the binding kinetics of intact Moloney murine leukemia retrovirus (MoMuLV) particles with NIH 3T3 cells was performed with an immunofluorescence flow cytometry assay. The virus-cell binding equilibrium dissociation constant (KD), expressed in terms of virus particle concentration, was measured to be 8.5 (+/- 6.4) x 10(-12) M at 4 degrees C and was three- to sixfold lower at temperatures above 15 degrees C. The KD of virus binding is about 1,000-fold lower than the KD of purified MoMuLV envelope. The association rate constant was determined to be 2.5 (+/- 0.9) x 10(9) M-1 min-1 at 4 degrees C and was 5- to 10-fold higher at temperatures above 15 degrees C. The apparent dissociation rate constant at 4 degrees C was 1.1 (+/- 0.4) x 10(-3) min-1 and was doubled for every 10 degrees C increase in temperature over the range tested (15 to 37 degrees C).     

Kinetic studies on Cro repressor-operator DNA interaction

The six operators of phage lambda and their consensus sequence were synthesized as 21 base-pair DNAs and their interactions with Cro repressor were studied using a filter binding assay. The measured equilibrium dissociation constants suggest that Cro has the highest affinity to the consensus operator (KD = 1.2 X 10(-12) M) and then the OR3 operator (KD = 2.0 X 10(-12) M), after that the affinity becomes lower in the following order: OR1, OL1, OL2, OL3, OR2. The competition experiments show that Cro forms the most stable complex with the consensus operator (t1/2 = 150 min), which is followed by the complex with OR3 (t1/2 = 70 min), OR1, OL1, OL2, OL3 and OR2. The association rate constants (ka) were also measured. They are approximately the same (2 X 10(8) to 4 X 10(8) m-1 s-1) for the consensus, OR3, OR2 and OR1 operators. These experiments have thus shown that the sequence difference in the operator affects the dissociation (KD and kd) but not the association (ka) process. The operators' binding strengths relative to OR1 are 14 (for consensus operator), 7.6 (OR3), 0.73 (OL1), 0.42 (OL2), 0.16 (OL3) and 0.1 (OR2). Seven different lengths of OR-containing DNA fragments were prepared. Measurement of kinetic parameters shows that the affinity of Cro to operator DNA (measured by KD) is essentially constant and independent of the DNA length, while the association and dissociation rate constants increase as the DNA length increases. This is consistent with the idea that Cro locates and leaves its operator via a two-step mechanism. It appears that Cro binds first at an arbitrary site on DNA, then is transferred to its operator site by a facilitated mechanism. The process is reversed when Cro dissociates from the operator. Most of our data fit to the theoretical expression formulated by Berg, Winter & von Hippel for the sliding mechanism. We conclude that Cro slides along the DNA to locate and leave the operator.     

Interaction of prothrombin with factor Va-phospholipid complexes

The effects of factor Va and the phospholipid-binding fragment of factor Va [factor Va light chain (LC), Mr 80000] on the binding of prothrombin, factor X, and factor Xa to phospholipid vesicles are reported. Equilibrium binding experiments were performed that utilized large-volume vesicles, which can be removed from the bulk solution by centrifugation. Factor Va decreased the dissociation constant of the prothrombin-phospholipid complex 50-fold, from 2.0 X 10(-7) M to 4.0 X 10(-9) M. For the factor X-phospholipid complex the decrease was 60-fold (1.8 X 10(-7) M to 3.0 X 10(-9) M) and for factor Xa, 160-fold (1.6 X 10(-7) M to 1.0 X 10(-9) M). The ratios of moles of protein bound to moles of total added factor Va at saturation of phospholipid-bound factor Va indicate an 1:1 stoichiometric complex of either factor Xa, factor X, or prothrombin and phospholipid-bound factor Va. In the presence of factor Va LC, the dissociation constants of factor Xa- and prothrombin-phospholipid complexes were increased, while the maximal protein-binding capacities of the vesicles were not affected by factor Va LC. The data suggest a competitive interaction between factor Xa and factor Va LC binding as well as between prothrombin and factor Va LC binding at the phospholipid surface. From this, it is concluded that the phospholipid-binding fragment of factor Va alone does not serve as the binding site for interactions of factor Xa and prothrombin with factor Va.     

Binding of N-[propionyl-3H]propionylated alpha-bungarotoxin and L-[benzilic-4,4'-3H] quinuclidinyl benzilate to CNS extracts of the cockroach Periplaneta americana

The nerve cord of the cockroach (Periplaneta americana) contains distinct saturable components of specific binding for the ligands N-[propionyl-3H]propionylated alpha-bungarotoxin and L-[benzilic-4,4'-3H]quinuclidinyl benzilate. N-[Propionyl-3H]propionylated alpha-bungarotoxin bound reversibly to homogenates with a Kd of 4.8 nM and Bmax of 910 fmol mg-1. The association rate constant (1.9 X 10(5) M-1 s-1) and dissociation rate constant (1.2 X 10(-4) s-1) yielded a Kd of 0.6 nM. Nicotinic ligands were found to displace toxin binding most effectively. The binding sites characterized in this way showed many similarities with the properties of the vertebrate neuronal alpha-bungarotoxin binding site. For a range of cholinergic ligands, inhibition constants calculated from toxin binding studies closely corresponded to their effectiveness in blocking the depolarizing response to acetylcholine recorded by electrophysiological methods from an identified cockroach motoneurone. The N-[propionyl-3H]propionylated alpha-bungarotoxin binding component therefore appears to be a constituent of a functional CNS acetylcholine receptor. Binding of L-[benzilic-4,4'-3H]quinuclidinyl benzilate was reversible with a Kd of 8 nM and Bmax of 138 fmol mg-1, determined from equilibrium binding experiments. The Kd calculated from the association rate constant (2.4 X 10(5) M-1 s-1) and dissociation rate constant (1.3 X 10(-4) s-1) was 1.9 nM. Muscarinic ligands were the most potent inhibitors of quinuclidinyl benzilate binding. The characteristics of this binding site resembled those of vertebrate CNS muscarinic cholinergic receptors. In contrast with vertebrate CNS, the nerve cord of Periplaneta americana contains more (approximately X 7) alpha-bungarotoxin binding sites than quinuclidinyl benzilate binding sites.     

Inhibition of glycogenolysis in isolated rat hepatocytes by the Rp diastereomer of adenosine cyclic 3',5'-phosphorothioate

The diastereomeric forms of adenosine cyclic 3',5'-phosphorothioate, Rp cAMPS and Sp cAMPS, were studied in isolated hepatocytes from fed rats for their ability to interact with the intracellular cAMP-dependent protein kinase and to affect the phosphorylase kinase-phosphorylase glycogenolytic cascade. Incubation of the cells with increasing concentrations of Sp cAMPS produced a concentration-dependent activation of cAMP-dependent protein kinase with a concomitant increase in the glycogenolytic rate. Half-maximal and maximal velocities of glycogenolysis were reached at 8 X 10(-7) and 1 X 10(-5) M Sp cAMPS, respectively. Incubation of the cells with 10(-9) to 10(-4) M Rp cAMPS had no effect on basal glucose production or on cAMP-dependent protein kinase activity. Incubation of the cells simultaneously with 3 X 10(-6) M Sp cAMPS and increasing concentrations of Rp cAMPS produced half-maximal inhibition of glycogenolysis at 1 X 10(-5) M Rp cAMPS and maximal inhibition at 1 X 10(-4) M. The concentrations of Sp cAMPS required for half-maximal and maximal activation of glycogenolysis were increased 10-fold when 1 X 10(-5) M Rp cAMPS was present. These data imply that Sp cAMPS is a cAMP-agonist while Rp cAMPS is a cAMP-antagonist.     

Affinity of hemoglobin for the cytoplasmic fragment of human erythrocyte membrane band 3. Equilibrium measurements at physiological pH using matrix-bound proteins: the effects of ionic strength, deoxygenation and of 2,3-diphosphoglycerate

The cytoplasmic fragment of band 3 protein isolated from the human erythrocyte membrane was linked to a CNBr-activated Sepharose matrix in an attempt to measure, in batch experiments, its equilibrium binding constant with oxy- and deoxyhemoglobin at physiological pH and ionic strength values and in the presence or the absence of 2,3-diphosphoglycerate. All the experiments were done at pH 7.2, and equilibrium constants were computed on the basis of one hemoglobin tetramer bound per monomer of fragment. In 10 mM-phosphate buffer, a dissociation constant KD = 2 X 10(-4)M was measured for oxyhemoglobin and was shown to increase to 8 X 10(-4)M in the presence of 50 mM-NaCl. Association could not be demonstrated at higher salt concentrations. Diphosphoglycerate-stripped deoxyhemoglobin was shown to associate more strongly with the cytoplasmic fragment of band 3. In 10 mM-bis-Tris (pH 7.2) and in the presence of 120 mM-NaCl, a dissociation constant KD = 4 X 10(-4)M was measured. Upon addition of increasing amounts of 2,3-diphosphoglycerate, the complex formed between deoxyhemoglobin and the cytoplasmic fragment of band 3 was dissociated. On the reasonable assumption that the hemoglobin binding site present on band 3 fragment was not modified upon linking the protein to the Sepharose matrix, the results indicated that diphosphoglycerate-stripped deoxyhemoglobin or partially liganded hemoglobin tetramers in the T state could bind band 3 inside the intact human red blood cell.