Dynamic mechanism of the self-assembly process of tobacco mosaic virus protein studied by rapid temperature-jump small-angle X-ray scattering using synchrotron radiation

The self-assembly process of tobacco mosaic virus protein (TMVP) was observed by rapid temperature-jump time-resolved solution X-ray small-angle scattering using synchrotron radiation. The temperature-jump device used for the X-ray measurements is rapid enough to cope with even the fastest-assembling process of TMVP, and accumulates data of reasonable signal-to-noise ratios with a minimum total counting time of 7.5 seconds. The measurements suggested that the 20 S disk of TMVP polymerized to stacked disks (short rods). The time to complete stacking varied from approximately 25 seconds to approximately 1200 seconds, depending on the solution condition and magnitude of the temperature gap. Higher protein concentration, ionic strength and temperature favoured faster association. The results were analysed in terms of a set of kinetic equations that describe the two-stage aggregation of TMVP with an equilibrium constant K1, and two rate constants k+2 and k-2 for association and dissociation of disks, respectively. The consistency of the analysis suggests that the TMVP assembly proceeds in two steps of: (1) the aggregation of A-proteins into double-layered disks; and (2) the stacking of double-layered disks. The kinetic analysis indicated that the stacking belongs to the lowest range of protein-protein interaction system.     

Determination of reaction volumes and polymer distribution characteristics of tobacco mosaic virus coat protein

A method that allows the quantitative determination of reaction volumes from sedimentation velocity experiments in an analytical ultracentrifuge is presented. Combined with a second method for detecting pressure-induced depolymerization, general characteristics of polymer distributions may be probed. We show that it is possible to determine if a sample is in an equilibrium or metastable state of subunit association. Our approach to probe macromolecular aggregation systems by small pressure perturbations is not restricted to the use of centrifuges. This method has been applied to characterize certain aspects of the polymerization of tobacco mosaic virus coat protein (TMVP). There are at least two helical polymer conformations in RNA-free coat protein rods. The smaller, helix I, polymers are limited to sizes below about 70 subunits (four to five helical turns) and undergo some kind of cooperative conformational change before further subunits may be added indefinitely. In contrast to helix I, the larger helix II polymers occur as broader and skewed size distributions. Under moderately strong polymerization conditions, the equilibrium state can contain both types of helical rods. The reaction volume for the addition of trimers is -220 ml/mol for both types of helical polymers. These results are compared with the results of previous thermodynamic analyses of TMVP polymerization.     

Time-resolved solution X-ray scattering of tobacco mosaic virus coat protein: kinetics and structure of intermediates

The kinetics of assembly and disassembly of tobacco mosaic virus coat protein (TMVP) following temperature jumps have been studied by small-angle X-ray scattering and turbidimetry. The structures of the principal aggregates of TMVP oligomers (A protein), intermediate size (helix I) and large size helical rods (helix II), have been characterized by their average radii of gyration of thickness, cross section, and shape obtained from the corresponding regimes of the small-angle scattering pattern. This structural information was obtained within seconds after the temperature-induced initiation of either polymerization or depolymerization and allowed us to detect transient intermediates. This methodology made it possible to observe and characterize the structure of a principal intermediate. Taken together with other kinetic information, these data suggest that polymerization of TMVP under virus self-assembly conditions may proceed via a single-layered helical nucleus that contains about 20 subunits. Previous studies have shown that overshoot polymerization of TMVP can occur and results in metastable long helical viruslike rods which subsequently depolymerize and then form short helical rods, depending on the conditions of the final equilibrium state. The longer rods (helix II) are overshoot polymers which form within seconds and contain 17 1/3 subunits per turn (helix IIB), in contrast to the subunit packing arrangement of 16 1/3 subunits per turn found in the shorter helical rods (helix IA). The latter packing arrangement is the one found in TMV. An overall polymerization scheme is proposed for the formation of these two helical forms of TMVP.     

Interaction of tobacco mosaic virus and tobacco mosaic virus protein with bovine serum albumin.

Bovine serum albumin (BSA) causes tobacco mosaic virus (TMV) to crystallize at pH values where both have negative charges. The amount of albumin required to precipitate the virus varies inversely with ionic strength of added electrolyte. At pH values above 5, the precipitating power is greatest when BSA has the maximum total, positive plus negative, charge. Unlike early stages of the crystallization of TMV in ammonium sulfate-phosphate solutions, which can be reversed by lowering the temperature, the precipitation of TMV by BSA is not readily reversed by changes in temperature. The logarithm of the apparent solubility of TMV in BSA solutions, at constant ionic strength of added electrolyte, decreases linearly with increasing BSA concentration. This result and the correlation of precipitating power with total BSA charge suggest that BSA acts in the manner of a salting-out agent. The effect of BSA on the reversible entropy-driven polymerization of TMV protein (TMVP) depends on BSA concentration, pH, and ionic strength. In general, BSA promotes TMVP polymerization, and this effect increases with increasing BSA concentrations. The effect is larger at pH 6.5 than at pH 6. Even though increasing ionic strength promotes polymerization of TMVP in absence of BSA, the effect of increasing ionic strength from 0.08 to 0.18 at pH 6.5 decreases the polymerization-promoting effect of BSA. Likewise, the presence of BSA decreases the polymerization-promoting effect of ionic strength. The polymerization-promoting effect of BSA can be interpreted in terms of a process akin to salting-out. The mutual suppression of the polymerization-promoting effects of BSA and of electrolytes by each other can be partially explained in terms of salting-in of BSA.     

Disk aggregates of tobacco mosaic virus protein in solution: electron microscopy observations

Previous studies of the coat protein of tobacco mosaic virus (TMVP) have shown that TMVP presumably exists as linear stacks of two-ring cylindrical disks in the 0.7 M ionic strength buffer used for crystallizing the disks for X-ray diffraction studies [Raghavendra, K., Adams, M.L., & Schuster, T.M. (1985) Biochemistry 24, 3298-3304]. The spectroscopic and sedimentation studies of solutions of TMVP under these crystallizing conditions have demonstrated a long-term metastability of these disk aggregates when they are placed in 0.1 M ionic strength buffers, as are used for reconstituting tobacco mosaic virus from TMVP and viral RNA. The present work describes an electron microscopic study of TMVP disk aggregates under the same solution conditions employed in the previous spectroscopic and sedimentation studies. The results show that in the pH 8.0 0.7 M ionic strength crystallization buffer TMVP exists as stacks of disks which range in size from about 6 to 24 layers, corresponding to 3-12 2-layer disk aggregates having 17 subunits per layer. These TMVP aggregates persist in a metastable form in 0.1 M ionic strength virus reconstitution buffer with no apparent changes in structure of the stacked disks. The results are consistent with the conclusions of the solution physical-chemical studies which suggest that the disk structure may not be related to the 20S TMVP aggregate that is the nucleation species in virus     

Uptake and incorporation of inositol by preimplantation mouse embryos.

The uptake of myo-inositol by preimplantation mouse embryos was investigated using [3H]myo-inositol. Uptake increased about 12-fold between one- and two-cell stages and increased again at the blastocyst stage (> 6-fold compared with the two-cell stage). Uptake at the blastocyst stage was time and temperature dependent; it was stimulated by sodium, inhibited by glucose and appeared to take place mainly via a saturable mechanism. Uptake in the presence of 6.25 mmol inositol l-1 was 1424 fmol inositol per blastocyst per h. About 10% of the [3H]inositol taken up by blastocysts during 8 h in culture was incorporated into lipid. Thin layer chromatography of the lipid showed that most of this inositol was incorporated into lipid material co-migrating with phosphatidylinositol with a small proportion co-migrating with phosphatidylinositol 4-phosphate.     

Ethylene Inhibition of Phytochrome-Induced Germination in Potentilla norvegica L. Seeds

Germination of Potentilla norvegica L. (rough cinquefoil) seeds stimulated by fluorescent irradiations of nearly 24 hours was inhibited by ethylene at <1 microliter per liter. Sensitivity to ethylene inhibition was highest during and immediately after the irradiation. By delaying ethylene treatment until about a day after the light potentiation, seeds escaped the inhibition. Ethylene inhibition may be readily reversed upon release of the gas and reirradiation of the seeds. Imbibition of seeds at 10 or 15°C, or at high temperatures of 35 and 40°C, partially prevented subsequent inhibition by ethylene. Alternating temperatures during germination nearly overcame the inhibition from 1 microliter per liter ethylene, but not higher doses. With brief red-irradiation and alternating temperatures, 0.1 microliter per liter ethylene promoted germination about 2-fold. These data suggest that ethylene may loosely associate on a site required for phytochrome action. The effect of temperature that opposed the inhibition may be to deny the association of ethylene with the site. Loose association is supported by the reversal of inhibition by gas release and increased temperature during germination. A blocking effect was shown by the failure of phytochrome to act when ethylene was present.     

In vivo-induced suppression of T cell proliferation: the relationship between the specificity of induction and control

We have previously shown that sc immunization of C57BL/10 (H-2b) mice with the tobacco mosaic virus protein (TMVP) or with its tryptic peptide number 8, representing residues 93-112 of TMVP, induces T cells which proliferate in vitro in response to TMVP and to peptide 8. In contrast, immunization of B10.BR (H-2k) mice either with TMVP or with peptide 8 induces T cells which respond in vitro to the homologous but not the heterologous Ag. In the present article , we report that in the B10.BR (H-2k) strain, ip prepriming with (TMVP) 7 days prior to sc immunization with peptide 8 causes a drastic reduction in the in vitro proliferative response of peptide 8-specific T cells while no such effect is seen in the congenic C57BL/10 (H-2b) strain. This suppression of T cell responsiveness can be transferred with TMVP-primed spleen cells. Moreover, deleting T cells from the transferred spleen cells abrogates the suppressive effect. In both H-2 haplotypes, ip prepriming with peptide 8 causes suppression of the proliferative T cell response induced by subsequent immunization with peptide 8. This prepriming has no effect on the response to TMVP immunization of B10.BR mice but does effect the response of C57BL/10 mice. Using various synthetic peptides to analyze the specificity of the suppression, we have determined that (1) T cells involved in the suppression of the proliferative T cell response to a single peptide determinant do not suppress the proliferative T cell response to other determinants on the protein antigen and (2) these T cells with suppressor function, and proliferating T cells which are ultimately regulated, can exhibit specificity for the same epitope. These studies suggest that there may exist fundamental differences as to how T cells which participate in suppression an proliferating T cells (which include mainly T helper cells) recognize protein antigens.     

Functional heterogeneity of memory B lymphocytes: in vivo analysis of TD-primed B cells responsive to secondary stimulation with TD and TI antigens

The functional heterogeneity of memory B cells induced by a single determinant, consisting of a decapeptide representing amino acid residues 103-112 of tobacco mosaic virus protein (TMVP), was analyzed. Decapeptide specific antibodies were elicited in mice adoptively transferred with TMVP-immune spleen cells when challenged with TMVP, decapeptide conjugated to succinylated human gamma-globulin (SHGG), or decapeptide conjugated to Brucella abortus (BA). Whereas secondary stimulation by either TMVP or decapeptide-SHGG was dependent on appropriately primed T cells, stimulation by decapeptide-BA was independent of conventional T cell help. Furthermore, memory B cells responsive to TMVP (TD), decapeptide-SHGG (TD), or decapeptide-BA (TI. 1 prototype) were shown to consist of overlapping populations because adoptive recipients of TMVP-primed cells challenged simultaneously with TD and TI decapeptide antigens did not result in a higher antibody response than that elicited by one of the TD antigens injected alone. However, decapeptide-BA consistently induced a smaller antidecapeptide response than either TMVP or decapeptide-SHGG. This suggested that only a fraction of the memory B cell population which was activated by the original priming antigen (thymus-dependent) was also responsive to secondary in vivo stimulation by the priming hapten conjugated to Brucella abortus. Detailed analyses of the antibodies induced in the recipients of TMVP-immune spleen cells after secondary challenge with either TMVP, decapeptide-SHGG, or decapeptide-BA failed to distinguish between the responsive memory B cells; the antidecapeptide antibodies induced by all three immunogens shared the same fine specificities and immunoglobulin isotype composition. These data are viewed as further evidence that subsets of TD-primed B cells, which may display differential sensitivity to cross-stimulation with TD and TI forms of the antigen, represent distinct stages of memory B cell maturation within a common B cell lineage. In support of this conclusion, we establish a developmental relationship between TI and/or TD responsive decapeptide memory B cell in the following communication.     

Structural aspects of a protein epitope and their role in the major histocompatibility complex control of T cell responsiveness.

We have previously shown that immunization of C57BL/10 (H-2b) mice with the tobacco mosaic virus protein (TMVP) or with its tryptic peptide number 8, representing residues 93-112 of TMVP, induces T cells which proliferate in vitro in response to TMVP and peptide 8. In contrast, immunization of congenic B10.BR (H-2k) mice with either TMVP or with peptide 8 induces T cells which respond in vitro to the homologous but not the heterologous antigen. The capacity to exhibit cross-reactivity between TMVP and peptide 8 on the T cell level has been shown to be under major histocompatibility complex (MHC)-linked genetic control. The lack of cross-reactivity has been attributed to the inability of the H-2k APC to present the appropriate epitope to T cells. In the present paper, we report results of a comparative analysis of the role of structural aspects of the epitope on the proliferative T cell responses from TMVP and peptide 8-immune C57BL/10 (H-2b) and B10.BR (H-2k) mice. Utilizing a panel of synthetic peptides representing portions of peptide 8 and a panel of peptide-protein conjugates, we have determined that peptide 8-immune T cells of the H-2k strain appear to recognize a single epitope within peptide 8, located at its N-terminus. In contrast, in the H-2b strain, both TMVP and peptide 8-immune T cells appear to recognize two overlapping epitopes within peptide 8; one located in the middle region and the other toward the N-terminus. Experiments with H-2b T cells revealed that random amino acids added to the carboxyl or amino-terminus of nonstimulatory peptides can confer activity to these peptides, demonstrating limited specificity of interaction between antigen and Iab. Results of experiments dealing with fixation of antigen-presenting cells suggest that TMVP requires processing in order to be recognized by peptide 8-immune H-2b proliferative T cells whereas peptide 8 does not. Taken together the results suggest that the T cell responsiveness to TMVP and peptide 8 exhibited by these two congenic strains H-2b and H-2k is not only controlled by the strains MHC but is also influenced by antigen processing. Antigen processing may eliminate a potential epitope for the primary induction and the secondary stimulation of B10.BR T cells.     

Thermodynamic analysis of the interaction of prolactin with its receptor in the rabbit mammary-gland microsomes.

The interaction of prolactin (PRL) with its membrane receptor depends markedly on temperature. Thermodynamic parameters for this reaction have been evaluated from data for time-course kinetics and equilibrium binding at multiple temperatures between 19 and 31 degrees C. The free-energy change with temperature and the van't Hoff plot were found to be linear. These suggest that there are minimal structure changes at the PRL-receptor contact site over this temperature range. The positive signs of the entropy and enthalpy of reaction, and of the entropy of activation (delta S++) for association, indicate that the hydrophobic bonding is the most significant force involved in PRL-receptor formation. The delta S++ for dissociation was negative, and the enthalpy of activation for dissociation was about 20.3 kJ.mol-1 larger than that for association, indicating that the PRL-receptor complex is further stabilized by contributions of hydrogen bonds and van der Waals contacts after the initial interaction. The free energy of activation for dissociation, at 25 degrees C was about 2.5-fold larger than that for association. This would cause slow dissociation of PRL from its receptor.     

Effects of the presence of an aldehydic abasic site on the thermal stability and rates of helix opening and closing of duplex DNA.

The presence of an abasic site in duplex DNA lowers the thermodynamic stability, as monitored by the optical melting temperature, and decreases the rate of imino proton exchange with water, by about an order of magnitude, as monitored by direct measurement of both the exchange lifetimes and the imino proton T1S. The exchange lifetimes of the imino protons with water as a function of base catalyst concentration were analyzed to determine the origin of the effect of the abasic site on imino exchange lifetimes. Analysis of the results showed that the helix opening rate is not significantly changed by the presence of an abasic site. The differences in exchange lifetimes are attributed to a faster helix closing rate in the presence of an abasic site. The faster rate of helix closing may be an important contribution to the stability of abasic sites in duplex DNA to base-catalyzed elimination reaction. It is noted that duplex DNAs containing analogues of the aldehydic abasic site apparently do not exhibit these exchange lifetime effects.     

Interconversion between glycogen and inositol in hibernating adults of a phytophagous ladybeetle,Epilachna vigintioctomaculata

The accumulation of inositol in hibernating adults of a phytophagous ladybeetle, Epilachna vigintioctomaculata, was demonstrated by a gas-chromatography and mass-spectrometry system. Inositol began to increase at the beginning of hibernation in early October and attained its maximum level of about 30 mg/g of body weight in January. It was also demonstrated that the source of inositol accumulated during hibernation is stored glycogen, and that interconversion between inositol and glycogen took place at the beginning and the end of hibernation.     

The Relationship between myo-Inositol, ATP and Rotational Streaming

The rotational streaming of cytoplasm in barley root hairs has been stimulated about 1.3–1.8 times through continuous treatment with various solutions of myo-inositol. The stimulation attained the same level as after ATP administration and was dependent on the external myo-inositol supply with the employed concentrations. The stimulation was cut off by simultaneous treatment with myo-inositol and uranyl salts. By using uranyl acetate the rate of streaming was maintained about the value of the control. The uranyl chloride caused an inhibition in the rotational streaming and later made it to cease altogether. The simultaneous treatment with myo-inositol and 2.4-DNP (dinitrophenol) induced a quick inhibition in 60% of the root hairs. consecutively stopping rotational streaming. It is assumed that the stimulation of rotational streaming is not due to the direct effect of myo-inositol but to ATP formed in the reaction: inositol hexaphosphate ++ ADP ? ATP + inositol. According to the results obtained by testing with uranyl salts, the phosphorylation of inositol probably takes place at the cell surface. The effect of 2,4-nNP points to the presence of two competitive metabolic processes involved in ATP consumption: the upkeep of rotational streaming and the uptake of substances by the plant cell.     

Temperature dependence of the structure of aggregates of tobacco mosaic virus protein at pH 7.2. Static synchrotron small-angle X-ray scattering

The small-angle X-ray scattering (SAXS) method using a synchrotron radiation source was applied to the study of the self-aggregation process of tobacco mosaic virus protein (TMVP) at a concentration of 5.0 or 12.0 mg ml-1 in 50 mM or 100 mM-phosphate buffer (ionic strengths approx. 0.1 and 0.2, respectively) at pH 7.2 in the temperature region of 4.8 to 25.0 degrees C. This paper presents the results of static measurements of SAXS. Sedimentation velocity experiments were performed simultaneously under the same conditions. These results are qualitatively parallel to those of the SAXS measurements, although the size of stacked disks derived from the SAXS measurements is larger than that derived from the sedimentation experiments, suggesting a change in the equilibrium conditions in the centrifugal field. Qualitative analysis of the SAXS data with model simulation calculations implies that the aggregation of TMVP consists of two steps: (1) the aggregation of A-protein comprising a few subunits to form double-layered disks; and (2) the random polymerization of double-layered disks by disk-stacking. Increase in temperature, ionic strength or protein concentration induced TMVP to polymerize to form a double-layered disk or a quadruple-layered short rod with consumption of A-proteins, accompanied by a small number of multi-layered short rods. The SAXS results indicate that the A-protein and the multilayered short rods are polydisperse with respect to size and shape, i.e. the mixture of A-protein, double-layered disks and multi-layered short rods coexists in the equilibrium state without pressure-induced partial dissociation of TMPV as observed during normal ultracentrifugation, and even under solution conditions in which the formation of double-layered disks or higher-order aggregates is favored.     

Isolation of a phosphomonoesterase from human platelets that specifically hydrolyzes the 5-phosphate of inositol 1,4,5-trisphosphate

Platelets, and a variety of other cells, rapidly hydrolyze the phosphoinositides in response to stimulation by agonists. One of the products of hydrolysis of phosphatidylinositol 4,5-diphosphate is inositol 1,4,5-trisphosphate, which recently has been suggested to mediate intracellular Ca2+ mobilization. We have found that human platelets contain an enzyme that degrades inositol 1,4,5-trisphosphate. We have isolated this soluble enzyme and find that it hydrolyzes the 5-phosphate of inositol 1,4,5-trisphosphate (Km = 30 microM, Vmax = 5.3 microM/min/mg of protein). The products of the reaction are inositol 1,4-diphosphate and phosphate. The apparent molecular weight of the enzyme is 38,000 as determined both by gel filtration and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence and absence of 2-mercaptoethanol. This enzyme is specific for inositol 1,4,5-trisphosphate. Other water soluble inositol phosphates as well as phosphorylated sugars are not hydrolyzed, while the only inositol containing phospholipid hydrolyzed is phosphatidylinositol 4,5-diphosphate at a rate less than 1% that for inositol 4,5-trisphosphate. The inositol 1,4,5-trisphosphate 5-phosphomonoesterase requires Mg2+ for activity and is inhibited by Ca2+, Ki = 70 microM. Li+, up to 40 mM, has no effect on enzyme activity. The duration and magnitude of any inositol 1,4,5-trisphosphate response in stimulated platelets may be determined by the activity of this enzyme.     

Acceleration of tetramer formation by the binding of inositol hexaphosphate to hemoglobin dimers.

The aggregation of deoxyhemoglobin dimers was studied by dropping the pH of a dilute solution of deoxyhemoglobin originally at high pH. In the presence of inositol hexaphosphate, a sharp increase in the rate of dimer association was observed. At higher concentrations of the phosphate, the rate decreased to a value close to that seen in the absence of phosphate. These observations require that inositol hexaphosphate binds to deoxyhemoglobin dimers. The dependence of the aggregation rate on phosphate concentration occurs because the reaction of a dimer containing bound phosphate with a phosphate-free dimer is 30 to 50 times faster than either the association of phosphate-free dimers or the association of dimers both containing bound phosphate.     

Inhibition by veratridine of carbachol-stimulated inositol tetrakisphosphate accumulation in rat brain cortical slices

The present studies examined the inhibitory effect of veratridine (a Na⁺ channel activator) on carbachol (a cholinergic agonist) stimulated inositol 1,3,4,5-tetrakisphosphate accumulation in rat brain cortical slices. Veratridine inhibited carbachol stimulation of inositol 1,3,4,5-tetrakisphosphate formation (after a delay of about 30 seconds) at 60 or 120 seconds when there was little inhibition of inositol 1,4,5 trisphophate accumulation. The inhibitory effect of veratridine on carbachol stimulated inositol 1,3,4,5-tetrakisphosphate accumulation was abolished in the presence of ouabain or tetrodotoxin but was unaffected in low calcium conditions. Veratridine reduced the total ATP content and this effect was abolished by tetrodotoxin. The inhibitory effect of 10 but not 30 M veratridine on inositol 1,3,4,5-tetrakisphosphate accumulation in the presence of carbachol was reversed by the presence of exogenous 8-bromo cyclic AMP or forskolin which activates adenylylcyclase. However, the decrease in brain slice ATP seen in the presence of veratridine was unaffected by forskolin. Our results are compatible with the hypothesis that veratridine inhibition of carbachol-stimulated inositol 1,3,4,5-tetrakisphosphate formation is due to depletion of ATP at the site of Ins 1,3,4,5-P₄ formation from Ins 1,4,5-P₃.Abbreviations used Ins 1,4,5-P₃ inositol 1,4,5 trisphosphate - Ins 1,3,4,5-P₄ inositol 1,3,4,5-tetrakisphosphate - PMA phorbol 12-myristate 13-acetate     

The association reaction between hemoglobin and carbon monoxide as studied by the isolation of the intermediates. Implications on the mechanism of cooperativity.

The concentrations of the intermediates in the association reaction between human hemoglobin and CO at 20 degrees C, pH 7, under conditions of negligible dissociation of the ligand, were measured by cryogenic techniques. The monoligated species were predominant at all values of overall ligand bound studied. The analysis of the experimental data assuming a scheme of four consecutive reactions indicated that the binding rates increased in a continuous fashion. A significant acceleration after the binding of the second molecule of ligand occurred in the presence of 0.1 M KCl, but not with the addition of an excess of inositol hexaphosphate, indicating that major functional, and possibly structural, transitions occur at the diligated state. Differences in the concentrations of the intermediates in the same state of ligation were observed under all conditions. The analyses of the data on the basis of schemes of multiple pathways of reaction indicated that the beta subunits reacted about 1.5 times faster than the alpha subunits in the first ligation reaction. After the addition of inositol hexaphosphate, the alpha subunits reacted about 1.5 times faster than the beta subunits in the first ligation step, but the overall rate of the first CO binding step was unchanged.     

Aggregation of deoxyhemoglobin subunits.

The formation of deoxyhemoglobin was examined by measuring the heme spectral change that accompanies the aggregation of isolated alpha and beta chains. At low hemeconcentrations (less than 10(-5) M), tetramer formation can be described by two consecutive, second order reactions representing the aggregation of monomers followed by the association of alphabeta dimers. At neutral pH, the rates of monomer and dimer aggregation are roughly the same, approximately 5 X 10(5) M(-1) X(-1) at 20 degrees. Raising or lowering the pH results in a uniform decrease of both aggregation rates due presumably to repulsion of positively charged subunits at acid pH and repulsion of negatively charged subunits at alkaline pH. Addition of p-hydroxymercuribenzoate to alpha chains lowers the rate of monomer aggregation whereas addition of mercurials to the beta subunits appears to lower both the rate of monomer and the rate of dimer aggregation. At high heme concentrations (greater than 10(-5) M) or in the presence of organic phosphates, the rate of chain aggregation becomes limited, in part, by the slow dissociation of beta chain tetramers. In the case of inositol hexaphosphate, the rate of hemoglobin formation exhibits a bell-shaped dependence on phosphate concentration. When intermediate concentrations of inositol hexaphosphate (approximately 10(-4 M) are preincubated with beta subunits, a slow first order time course is observed and exhibits a half-time of about 8 min. As more inositol hexaphosphate is added, the chain aggregation reaction begins to occur more rapidly. Eventually at about 10(-2) M inositol hexaphospate, the time course becomes almost identical to that observed in the absence of phosphates. The increase in the velocity of the chain aggregation reaction at high phosphate concentrations suggests strongly that inositol hexaphosphate binds to beta monomers and, if added in sufficiently large amounts, promotes beta4 dissociation. A quantitative analysis of these results showed that the affinity of beta monomers for inositol hexaphosphate is the same as that of alphabeta dimers. Only when tetramers are formed, either alpha2beta2 or beta4, is a marked increase in affinity for inositol hexaphosphate observed.