Stacking self-association of pyrimidine nucleosides and of cytosines: effects of methylation and thiolation.

Stacking self-association equilibria in aqueous solutions of m3uridine, m34,2',3',5'uridine, 2'-deoxyuridine, m13,4,4cytosine, m14,4,4,5cytosine, s2cytidine and s4thymidine were studied at various temperatures by vapour-pressure osmometry. Equilibrium constants Kst's were computed on the assumption of the isodesmic model of self-association. Enthalpies of association were also obtained from the temperature dependence of Kst according to the van't Hoff equation. Analysis of the equilibrium and thermodynamic parameters demonstrated involvement of hydrophobic interactions in the stabilization of complexes of tetramethyluridine. Dipole-induced dipole interactions seem to predominate in the formation of s2C, s4T and of both dimethylaminocytosine complexes.     

Self-association of rabbit muscle phosphofructokinase at pH 7.0: stoichiometry

The self-association of rabbit muscle phosphofructokinase at pH 7.0 was investigated by velocity sedimentation. The process was demonstrated to be in a rapid, dynamic equilibrium. The concentration dependence of the weight-average sedimentation coefficient was monitored within the range of 10-750 microgram/mL. The sedimentation properties of phosphofructokinase were analyzed by theoretical simulations or an associating system in rapid equilibrium. In the absence of any ligands and at a temperature of 23 degrees C, the simplest computed model which gives the best fit between theoretical and experimental points can be described as progressive association of monomer in equilibrium or formed from tetramer in equilibrium or formed from 16-mer with apparent equilibrium constants K4 = 5.06 X 10(5) (mL/mg)3 and K16 = 3.25 X 10(23) (mL/mg)15. However, at 5 degrees C, the equilibrium was altered and can best be described as monomer in equilibrium or formed from dimer in equilibrium or formed from tetramer in equilibrium or formed from 16-mer.     

The self-association of human spectrin at high concentration.

The self-association of purified human spectrin has been studied at sedimentation equilibrium over a wide range of concentration (0-20 g/L) at 30 degrees C and pH 7.5. Coincidence of apparent weight average molecular weight and omega (r) plots as a function of total spectrin concentration indicated that equilibrium was attained and that no significant concentration of solute was incapable of participating in the self-association reaction. Under these conditions, no significant dissociation of the heterodimer to component polypeptide chains could be detected. The behavior of spectrin between 0 and 20 g/L can be described reasonably well by a cooperative isodesmic model, in which the protomer for association is the alpha beta heterodimer. With this model, the equilibrium constant for the heterodimer-tetramer step, K24, is 2 x 10(6) M-1, and K(iso), the equilibrium constant describing all other steps, is approximately 0.2 x 10(6) M-1. The returned value of the second virial coefficient for this model, 1.0 x 10(-7) L mol g-2, is consistent with the lower limit of values calculated for the heterodimer from the charge and Stokes radius of spectrin. On the other hand, the attenuated indefinite association model fails to describe the self-association of spectrin adequately over the range 0-20 g/L. Systematic decreases in the estimates of the second virial coefficient and the equilibrium constants for association beyond the tetramer suggest that the assumption of a single value of the second virial coefficient may not be appropriate for spectrin, and that non-ideality would best be taken into account by consideration of the detailed solution composition.     

Quaternary interactions in hemoglobin beta subunit tetramers. Kinetics of ligand binding and self-assembly

We have investigated the rates of monomer in equilibrium with tetramer self-association of oxygenated beta SH subunits of human hemoglobin A as well as the influence of self-association on the binding kinetics for O2 and CO. A 4 beta in equilibrium with 2 beta 2 in equilibrium with beta 4 assembly pathway can be used to describe the association equilibria and kinetics. We have determined all four elementary rate constants for this assembly pathway at 15 degrees C in 0.1 M Tris-HCl, 0.1 M NaCl, 1 mM Na2EDTA, pH 7.4. These data imply that a significant amount (approximately 17%) of beta 2 can be present. Laser photolysis kinetic studies of O2 binding indicate that the O2 association rate constant is unaffected by the degree of self-association. In contrast, photolysis of beta CO solutions shows an overall rate of CO binding that increases at higher protein concentrations. These data are consistent with a concentration-dependent equilibrium between two protein species with CO association rates differing by a factor of 2.5, but they do not appear to be compatible with a direct assignment of different CO binding rates to the different assembly states. Rather, we believe the data imply that CO binding to beta oligomers is heterogeneous, with both a fast binding and a slow binding form being present in single association states. The fast binding form predominates (approximately equal to 87%) in beta 4, while the beta monomer has very little or none of the fast binding form. We propose that the slow binding component within beta 4 may be those subunits with rotationally disordered hemes (La Mar, G. N., Yamamoto, Y., Jue, T., Smith, K. M., and Pandey, R. K. (1985) Biochemistry 24, 3826-3831). The implications of these findings for the use of isolated subunits as models for the subunits within "R state" hemoglobin tetramers are discussed.     

Reversible self-association of a human myeloma protein. Thermodynamics and relevance to viscosity effects and solubility

Monoclonal IgG paraproteins associated with multiple myeloma, Felty's syndrome, and idiopathic cryoglobulinemia frequently produce disease due to a tendency to self-associate in vivo. The insolubility and viscosity effects of these proteins are of specific interest as molecular disease mechanisms. In sedimentation equilibrium studies at 21 degrees C an IgG1-lambda myeloma protein (IgG-MIT) associated with the hyperviscosity syndrome is shown to undergo a reversible polymerization reaction. On the basis of the theory and data-fitting methods of Adams and co-workers [Tang, L. H., Powell, D. R., Escott, B. M., & Adams, E. T., Jr. (1977) Biophys. Chem. 7, 121-139], the data are consistent with a nonideal cooperative indefinite (SEK type III) model self-association in which one equilibrium constant (K12 = 6.3 X 10(3) L/m) governs dimerization while another (K = 1.7 X 10(4) L/m) governs all subsequent additions of monomer to the polymer. Temperature effects on K12 and K between 11 and 30 degrees C suggest negative van't Hoff enthalpies for all association steps and a positive entropy change [delta S degree = 2.5 cal/(mol-deg)] for steps beyond the dimer. An increase in ionic strength from I = 0.03 to I = 0.18 promotes the polymerization of IgG-MIT through a marked increase in K while paradoxically enhancing bulk solubility. These results suggest that this self-association proceeds through a combination of weak nonionic and hydrophobic interactions. The enhancement of both polymerization and solubility by increased ionic strength suggests that the hyperviscosity induced by IgG-MIT results from its ability to form large, highly soluble polymers in serum.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ionization and self-association of unconjugated bilirubin, determined by rapid solvent partition from chloroform, with further studies of bilirubin solubility.

Our studies of equilibrium solubilization of crystals of unconjugated bilirubin (UCB) in buffered aqueous NaCl (1988. J. Lipid Res. 29: 335-348) suggested that the two carboxylic pKa values were 6.8 and 9.3 and the solubility of UCB diacid was 0.1 microM. These data, however, were not ideal, due to possible effects of crystal size, metastability, 96-h incubation times with formation of polar derivatives, impurities in the bilirubin, and imprecision of analyses at low concentrations of UCB ([UCB]). In the present study, designed to determine the pKa values and self-association of UCB, these problems were minimized by solvent partition of UCB from solution in CHCl3 into buffered aqueous NaCl. There was no crystal phase. Equilibrium was attained rapidly (10 min); UCB and CHCl3 were highly purified; and accurate diazo assay of low [UCB] in the aqueous phase, [Bw], was achieved by concentrating the UCB through back-extraction into a small volume of CHCl3. By determining effects on partition rations of varying the [UCB] in the CHCl3 phase, [Bc], we could assess also the self-association of UCB species in the aqueous phase. Partition ratios (P = Bw/Bc) did not differ between initial and repeat extractions, indicating insignificant concentrations of polar UCB derivatives. Similar P ratios were obtained when equilibrium was approached from a supersaturated aqueous phase. At 21-25 degrees C, mu = 0.15, the data (n = 76) fit the equation: log P = log Po + log[1 + 10(pH-A) + 10(2pH-B) + Bc.10(4pH-D)]; the bracketed terms reflect P for H2Bo (diacid), HB- (monoanion), B= (dianion), and (B=)2 dimer, respectively. Computer-fitted values for constants (+/- SD) were: Po = P for H2Bo = 5.79 x 10(-5); A = pK1 = 8.12 +/- 0.23; B = pK1 + pK2 = 16.56 +/- 0.10; pK2 = 8.44 +/- 0.33; D = pk22 + 2(pK1 + pK2) -log(2Po) = 37.64 +/- 0.07, and k22 = 0.26 microM-1 [formation constant of (B=)2 dimer]. In ancillary studies, multiple cycles of direct dissolution of UCB crystals revealed a progressive decrease in aqueous solubility of UCB as fine crystals were removed; this effect was minimal in CHCl3. Unlike in water, moreover, varied UCB crystal forms had similar solubilities in CHCl3, with [Bc] = 1.14 mM at saturation. As determined from [Bc]sat.Po, the aqueous solubility of H2Bo was 66 nM.(ABSTRACT TRUNCATED AT 400 WORDS)     

Temperature and pH dependence of the self-association of human spectrin

The self-association of human spectrin between 21 and 35 degrees C and between pH 6.5 and 9.5 has been studied at sedimentation equilibrium. For a given set of solution conditions between pH 6.5 and 8.5, coincidence of omega function plots as a function of total spectrin concentration (0-2 g/L) indicated that equilibrium was attained and that no significant concentration of solute was incapable of participating in the self-association reaction. Above pH 8.5, however, irreversible aggregation occurred, inferred from a failure of overlap in the omega function and molecular weight distributions. The behavior of spectrin can best be described by a cooperative isodesmic model, in which the promoter for association is the heterodimer and for which K12 is between 10(6) and 10(7) M-1 (depending on pH and temperature) and all other K are approximately 10(6) M-1. The returned values of the second viral coefficient for this model fall within the range calculated from the charge and Stokes radius of spectrin. Association appears to be favored slightly by decreased temperature and by decreased pH. The pH dependence resides only in K12 and is consistent with the presence of a single group, possibly histidine, displaying a slightly higher pKa value in the tetramer than in the dimer. The association reaction appears to be driven by the loss of enthalpy associated with release of strain in the heterodimer. The association sites appear to be conserved in the association reactions, consistent with the images from electron microscopy.(ABSTRACT TRUNCATED AT 250 WORDS)     

A thermodynamic model for the self-association of human spectrin

The self-association of human spectrin at 28.8 degrees C in 0.11 M salt (pH 7.5) has been studied by means of sedimentation equilibrium. Coincidence of omega function plots as a function of total spectrin concentration (0-2 g/L) indicated that equilibrium was achieved and that no significant concentration of solute was incapable of participating in the self-association reaction. On the basis of the root-mean-square deviation of the fits and the randomness of the residuals, the behavior can be described equally well, either by a cooperative isodesmic model, in which K12 approximately 2 x 10(6) M-1 and all other K approximately 10(6) M-1, or by an attenuated scheme in which K(i-1)i approximately (3.5 x 10(6)/i M-1. The returned values of the second virial coefficient, B, for both these models fall within the range calculated from the charge and Stokes radius of spectrin. A mechanism for spectrin self-association consistent with both schemes is proposed in which spectrin heterodimers undergo a reversible opening at the self-association interface. These open heterodimers then undergo indefinite self-association to form a series of open-chain oligomers in dynamic equilibrium with closed-loop oligomers.     

Studies on human serum high-density lipoproteins. Self-association of human serum apolipoprotein A-II in aqueous solutions.

Some of the solution properties of pure preparations of human serum high-density apolipoprotein A-II were studied by sedimentation equilibrium ultracentrifugation, conducted at different apoprotein concentrations and at several speeds. The concentration dependence of the apparent weight average molecular weight indicated that apolipoprotein A-II, when dissolved in 0.02 MEDTA (pH 8.6), undergoes self-association. Over a protein concentration range between 0.8 and 1.5 mg/ml, the self-association could best be described by a monomer-dimer-trimer step association, although indefinite self-association could not be ruled out. The equilibrium constants obtained were sufficient to describe the system over the concentration range investigated.     

The temperature-dependent self-association of beta-lactoglobulin C in glycine buffers

The self-association of β-lactoglobulin C at low pH (ca. 2.5) in glycine buffers has been studied at four temperatures, 10, 16, 20, and 25 °C, by low- and high-speed sedimentation equilibrium experiments. One buffer had an ionic strength of 0.1 and the other an ionic strength of 0.2. With either buffer the concentration dependence of the apparent weight average molecular weight, M_wa, was characteristic of a nonideal self-association. Like its genetic variants, β-lactoglobulin A and B, the self-association of β-lactoglobulin C increased with decreasing temperature; however, at the same temperature the association was always stronger in the buffer having the higher ionic strength. Several models were used to test the self-association, and a monomer-dimer self-association seemed to describe the self-association best with either buffer. Values of the association equilibrium constant, K₂, and the second virial coefficient, BM₁, are reported at each temperature for both series of experiments. Values of the thermodynamic functions, ΔG °, ΔH °, and ΔS °, are also reported for these experiments.     

Evidence for uptake of 8-methoxypsoralen and 5-methoxypsoralen by cellular nuclei

The fluorescent appearance of oral mucosa cells treated with 8-methoxypsoralen (8-MOP) and 5-methoxypsoralen (5-MOP) was observed by means of fluorescence microscopy. Fluorescence at the nuclei was weakened in 8-MOP-treated cells, while it was intensified in 5-MOP-treated cells. These findings were consistent with changes in the fluorescence intensities on association of the psoralen derivatives with DNA in aqueous solution. This intensity change of fluorescence and also the blue shift of the fluorescence maximum of the derivatives on association suggested that the environment around the psoralen molecules is as little polar as methanol. From the results of these fluorescence microscopic observations and spectroscopic analysis of fluorescence of derivatives interacting with DNA during equilibrium dialysis, we concluded that 8-MOP, as well as 5-MOP, is incorporated by nuclei of human cells.     

Stability and instability properties of aggregation of single chain amphiphiles into binary mixtures

In a lysophospholipid binary mixture, there are three ways of association between the mixture components of single-chain amphiphiles: (a) between two identical molecules each of the first and second component (self-association process) and (b) between two different molecules (cross-association process). Association probabilities for three binary mixtures were analysed as functions depending on the electric dipole moments of the polar head groups. A 3-D view representation is most suitable for this analysis. The most important finding is that for certain values of the electric dipole moments there are molecular couples which have a maximum stability to the changes in the external electrolytic medium. This fact confirms the formation of clusters and their stability, which is equivalent to the existence of micro-heterogeneities within the lipid bilayers. On the other hand, there are unstable molecular associations, and this fact influences the appearance of some phase transitions. Generally, the increase of the electric dipole moment or the increase of the acyl-chain length of one component from a binary lipid mixture decreases the self-association probability between its own molecules, but it increases the self-association probability of the other mixture components. Furthermore, the cross-association probability has high values for any binary lipid mixture of single-chain amphiphiles.     

Thermodynamical model of indefinite mixed association of two components and NMR data analysis for caffeine-AMP interaction

This paper describes the model used to estimate the parameters of caffeine-AMP interactions from corresponding 1H-NMR measurements and some methods of data analysis by which the applicability of the model has been checked. The model of mixed association is applicable to a mixture of any two substances A and C which exhibit indefinite aggregates in both self-association and mixed association. In aggregates, only nearest neighbour interaction is assumed. The model is described by three equilibrium constants: Kaa and Kcc (for self-association of A, or C, respectively), and Kac (for mixed association).     

Interaction of vinblastine with calf brain tubulin: multiple equilibria

The binding of the anticancer drug vinblastine to calf brain tubulin was measured by a batch gel filtration method in PG buffer (0.01 M NaPi, 10(-4) M GTP, pH 7.0) at three different protein concentrations. The Scatchard binding isotherms obtained were curvilinear. The binding of the first vinblastine molecule to each tubulin alpha-beta dimer (Mr 110,000) was enhanced by an increase in the protein concentration. Additional binding of vinblastine to the protein was independent of the protein concentration. Theoretical ligand binding isotherms were calculated for a ligand-induced macromolecule self-association involving various ligand stoichiometries and association schemes. Fitting of the experimental data to these isotherms showed that the system can be described best by a one-ligand-induced isodesmic indefinite self-association. The pathway giving the best fit consists of a ligand-mediated plus -facilitated self-association mechanism. The self-association-linked bound vinblastine binds specifically at a site with an intrinsic binding constant K1 = 4 X 10(4) M-1. Additional vinblastine molecules can bind less strongly to tubulin in probably nonspecific fashion, and the previous reports of two specific sites on alpha-beta tubulin for binding vinblastine are incorrect. The self-association constant K2 for liganded tubulin is 1.8 X 10(5) M-1. This analysis is fully consistent with the conclusions derived earlier from the linked function analysis of the vinblastine-induced tubulin self-association [Na, G. C., & Timasheff, S. N. (1980) Biochemistry 19, 1347-1354; Na, G. C., & Timasheff, S. N. (1980) Biochemistry 19, 1355-1365].     

Light-scattering studies of the alpha-ketoglutarate dehydrogenase complex from escherichia coli. I. Characterization of the self-association of the complex

The self-association of Escherichia coli alpha-ketoglutarate dehydrogenase complex (KGDC) purified by a column Chromatographic technique, was characterized by light-scattering photometry. The complex adopts a solution conformation somewhat larger than that observed in the electron microscope. The evidence suggests a nonideal indefinite self-association model for KGDC in KCl, phosphate buffer. The KGDC monomer has a molecular charge of about -3 x 10(2) at neutral pH. The self-association is promoted by increasing KCl concentrations, pH (in the range from 6.3 to 7.4) and temperature (from 20 to 30 degrees C). The effects of pH changes suggest a release of protons during the self-association and a minor 'preferential' interaction of phosphate ions. For the association of one monomer to the aggregate at neutral pH and 25 degrees C. DeltaG degrees = -7.8 kcal mol(-1). DeltaH degrees = 24 kcal mol(-1) and DeltaS degrees = 1.1 x 10(2) cal mol(-1) K(-1). These data indicate that hydrophobic interactions drive the association. Thermodynamically, the self-association of KGDC is a complex phenomenon and may serve to stabilize the enzyme complex in solution.     

Self-association of rabbit muscle phosphofructokinase: effects of ligands

The effects of ligands on the self-association of rabbit muscle phosphofructokinase (PFK) were investigated by velocity sedimentation at pH 7.0 and 23 degrees C. The concentration dependence of the weight-average sedimentation coefficient was monitored in the presence of these ligands. The mode of association and equilibrium constants characterizing each association step were determined by theoretical fitting of the sedimentation data. The simplest mode of association for the PFK system is M in equilibrium M2 equilibrium M4 in equilibrium M16. Ligands and temperature would perturb the various equilibrium constants without altering the mode of association. The apparent equilibrium constants for the formation of tetramer, K4app, are increased in the presence of 0.1 mM ATP and 1.0 mM fructose 6-phosphate. The value of the sedimentation coefficient for the tetramer, S4 degrees, that would best fit the data is 12.4 S instead of 13.5 S determined in the absence of substrates, thus implying a structural change in the tetramer induced by substrates. Only an insignificant amount of dimer is present under the experimental conditions. The presence of activators, ADP or phosphate, enhances the formation of tetramers, and S4 degrees assumes a value of 13.5 S. Similar results are obtained with decreasing concentrations of proton. The presence of the inhibitor, citrate, however, favors the formation of dimers. The equilibrium constants determined as a function of ADP concentration were further analyzed by the linked-function theory derived by Wyman [Wyman, J. (1964) Adv. Protein Chem. 19, 224--285], leading to the conclusion that the formation of a tetramer involves the binding of two additional molecules of ADP per monomer. Similar analysis results in a conclusion that the formation of a dimer involves the binding of one additional molecule of citrate per phosphofructokinase subunit.     

Self-association mode of a flavoenzyme D-amino acid oxidase from hog kidney. II. Stoichiometry of holoenzyme association and energetics of subunit association

The self-association pattern of D-amino acid oxidase holoenzyme in 0.1 M sodium pyrophosphate, pH 8.3, at 25 degrees C was examined by the low-angle laser light-scattering method. As to the results of nonlinear least-squares analysis of the apparent weight-average molecular weight (Mwapp) versus protein concentration (c) data, the following three models fitted equally well the data over the concentration range of 0.03-11.4 mg/ml: 1) the model of isodesmic indefinite self-association of the monomer where the dimerization constant differs from the isodesmic association constant, 2) the model which involves the dimerization of the monomer and isodesmic indefinite self-association of the dimer, and 3) the model which involves the trimerization of the monomer and isodesmic indefinite self-association of the trimer. In a more limited concentration range (0.3-11.4 mg/ml), a model of isodesmic indefinite self-association of the stable dimer where the dimer does not dissociate into the monomers cannot be excluded from the above three models. Measurements with the concentration range lowered to 0.03 mg/ml enabled us to exclude unequivocally the model involving such a stable dimer and to extrapolate the Mwapp data to the Mr of the monomer at infinite dilution as in the case of the apoenzyme. The observed sedimentation boundary profiles were qualitatively consistent with the idealized boundary profiles calculated with the model which involves the dimerization of the monomer and isodesmic indefinite self-association of the dimer, so this model is the most probable of the models examined. These results provide the first evidence that the association mode of the holoenzyme is different from that of the apoenzyme, i.e. isodesmic indefinite self-association of the monomer (Tojo, H., Horiike, K., Shiga, K., Nishina, Y., Watari, H., and Yamano, T. (1985) J. Biol. Chem. 260, 12607-12614). The overall linkage scheme, between binding of coenzyme FAD and subunit association, was considered, and the overall free energy change in each process in the scheme was calculated. The total stabilization energies of the intersubunit interaction in the holoenzyme relative to the apoenzyme were found to be -2.2 kcal/mol at the dimerization step and -0.5 kcal/mol at the step of the addition of the dimer to any 2i-mer (i = 1,2, ...).     

The self-association of chicken-erythrocyte histones.

The self-association of the separate histone fractions isolated from chicken erythrocytes has been studied in solution at a number of different pH values and ionic strengths. The apparent molecular weights of the histones were determined over a range of macromolecular concentrations using the techniques of osmotic pressure and sedimentation equilibrium. Histone F2c (H5) did not associate under any of the conditions investigated whereas the other histone fractions all appeared to undergo self-association forming dimers, dimers of dimers, etc. The degree of association increased with the pH and ionic strength of the medium. The tendency to aggregate increased in the order; histone F2c (H5) (non-aggregating), histone F2b (H2B), histone F2a2 (H2A), histone F3 (H3), histone F2a1 (H4) (highly aggregating). In the case of histone F2a2 (H2A) at pH 3.0 and ionic strength 0.1, the apparent weight-average molecular weight was determined at a number of macromolecular concentrations at five different temperatures. The self-association was analysed according to the method of Adams (published by Beckman Instruments Inc. in 1967) and shown to be a monomer-dimer-tetramer equilibrium. The association constants were evaluated at each of the temperatures studied and from their variation with temperature the values of the enthalpy and entropy of association were calculated. The intermolecular association was characterised by only a small change in enthalpy but a large, positive, change in entropy. This suggests that the association of histones at acid pH is due to hydrophobic interactions between the relatively uncharged segments of like polypeptide chains.     

Characterization of the cooperative cross-linking of doxorubicin N-hydroxysuccinimide ester derivatives to water soluble proteins.

Protein-anthracycline interactions have been examined by using reactive N-hydroxysuccinimide ester derivatives of doxorubicin. These compounds cross-link to lysine epsilon-amino groups with high efficiency and offer the possibility for structural studies of protein-anthracycline complex formation by using gel filtration, ultracentrifugation and spectrophotometric methods. The results are in accordance with association of anthracycline to the hydrophobic ligand binding cavities of serum albumin. The results for proteins not having hydrophobic domains (IgG, serum transferrin, lactotransferrin, ovotransferrin) suggest that complex formation is cooperative and involves two steps: initial self-association of anthracycline into aggregated structures and subsequent binding of protein at the aggregate surface. With serum transferrin, anthracycline self-association makes possible the assembly of stable nanometer-sized protein-anthracycline particles held together by non-covalent bonds. This reaction, which is highly reproducible and efficient, may have applications in the field of development of anthracycline carrier systems.     

The self-association of L-alpha hydroxyacid oxidase.

As the published values for the molecular weight of L-alpha-hydroxyacid oxidase vary from 89 000 to 430 000, it is possible that such variations could be due to a concentration dependence of the molecular weight. The molecular weight of rat L-alpha-hydroxyacid oxidase was studied over a wide range of concentrations, using equilibrium sedimentation and gel exclusion chromatography. The partial specific volumes (0.726 and 0.730 for hydroxyacid oxidase A and hydroxyacid oxidase B, respectively) were calculated from the amino acid compositions, and were used to calculat molecular weights from the equilibrium sedimentation data. The molecular weight at infinite dilution was found to be 150 000 for both the A and B isozymes. Both isozymes exhibit association-dissociation behaviour at low concentrations. The self-association of the hydroxyacid oxidase B isozyme can be described by the relation (see article) where K1,2 = 5.4-10(5) M-1 and K2,4 = 1.7-10(5) M-1. Previously published values of the molecular weight of these isozymes are in accord with the observed concentration dependence.