High-speed Sedimentation Equilibrium Research Articles

Allowance for effects of thermodynamic nonideality in sedimentation equilibrium distributions reflecting protein dimerization

This reexamination of a high-speed sedimentation equilibrium distribution for α-chymotrypsin under slightly acidic conditions (pH 4.1, IM 0.05) has provided experimental support for the adequacy of nearest-neighbor considerations in the allowance for effects of thermodynamic nonideality in the characterization of protein self-association over a moderate concentration range (up to 8mg/mL). A widely held but previously untested notion about allowance for thermodynamic nonideality effects is thereby verified experimentally. However, it has also been shown that a greater obstacle to better characterization of protein self-association is likely to be the lack of a reliable estimate of monomer net charge, a parameter that has a far more profound effect on the magnitude of the measured equilibrium constant than any deficiency in current procedures for incorporating the effects of thermodynamic nonideality into the analysis of sedimentation equilibrium distributions reflecting reversible protein self-association.

Solution Structure of Porcine Delta Sleep-inducing Peptide Immunoreactive Peptide A Homolog of the ShortsightedGene Product

The 77-residue delta sleep-inducing peptide immunoreactive peptide (DIP) is a close homolog of the Drosophila melanogaster shortsighted gene product. Porcine DIP (pDIP) and a peptide containing a leucine zipper-related partial sequence of pDIP, pDIP(9-46), was synthesized and studied by circular dichroism and nuclear magnetic resonance spectroscopy in combination with molecular dynamics calculations. Ultracentrifugation, size exclusion chromatography, and model calculations indicated that pDIP forms a dimer. This was confirmed by the observation of concentration-dependent thermal folding-unfolding transitions. From CD spectroscopy and thermal folding-unfolding transitions of pDIP(9-46), it was concluded that the dimerization of pDIP is a result of interaction between helical structures localized in the leucine zipper motif. The three-dimensional structure of the protein was determined with a modified simulated annealing protocol using experimental data derived from nuclear magnetic resonance spectra and a modeling approach based on an established strategy for coiled coil structures. The left-handed super helical structure of the leucine zipper type sequence resulting from the modeling approach is in agreement with known leucine zipper structures. In addition to the hydrophobic interactions between the amino acids at the heptade positions a and d, the structure of pDIP is stabilized by the formation of interhelical i to i' + 5 salt bridges. This result was confirmed by the pH dependence of the thermal-folding transitions. In addition to the amphipatic helix of the leucine zipper, a second helix is formed in the NH2-terminal part of pDIP. This helix exhibits more 310-helix character and is less stable than the leucine zipper helix. For the COOH-terminal region of pDIP no elements of regular secondary structure were observed.

Characterization of the ligand binding domain of human retinoid X receptor alpha expressed in Escherichia coli

In order to study the structural details of ligand protein interactions of the human retinoid X receptor alpha (hRXR alpha), the DEF and EF domains of the receptor were expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli. The fusion proteins were expressed at high levels and were affinity-purified by chromatography over glutathione-agarose. The DEF and EF domains were cleaved from the fusion proteins by digestion with thrombin. Retinoic acid binding was quantitated using two different methods. The apparent dissociation constant (Kd) and the stoichiometry of 9-cis-retinoic acid binding were performed by monitoring quenching of protein fluorescence. To directly compare the binding affinity of the E. coli-derived truncated hRXR alpha with full-length hRXR alpha expressed in transiently transfected COS cells, Scatchard analyses of [3H]9-cis-retinoic acid binding assays were performed. Both methods of analysis indicate that while the cleaved DEF peptide bound 9-cis-retinoic acid tightly, the cleaved EF peptide exhibited variable binding activity between preparations. By fluorimetric analysis, the Kd of the cleaved DEF peptide was estimated to be 3 +/- 0.5 nM with a stoichiometry of 1:1.1 +/- 0.1. By Scatchard analysis, the Kd values for [3H]9-cis-retinoic acid to the GST-hRXR alpha (DEF) peptide and the cleaved DEF peptide were estimated to be 1.8 nM and 5.6 nM, respectively. The estimated molecular mass from high speed sedimentation equilibrium experiments was 36 +/- 2 kDa for the apo-DEF peptide alone and 38 +/- 3 kDa for the holo-DEF peptide complexed with 9-cis-retinoic acid. This suggests that the recombinant ligand binding domain was predominantly in the monomer form. However, dimers of the cleaved DEF peptides were detected in chemical cross-linking experiments both in the presence and absence of 9-cis-retinoic acid. Since the purified E. coli-derived truncated hRXR alpha DEF peptide appears to fully retain its ligand binding activity, it should provide a useful model system for further structural analysis of ligand-protein interactions.

Evaluation of dead-end ultrafiltration properties by ultracentrifugation method.

In protein ultratiltration, solution environment factors such as pH and solvent density are important in controlling the filtration flux. Also, the properties of the filter cake of macromolecular solutes formed on the membrane surface play a vital role in determining the filtration flux in ultratiltration. On the basis of the sedimentation velocity technique and the high-speed sedimentation equilibrium technique using an analytical ultracentrifuge, the influence of the protein solution pH on the time variation of the filtration flux in unstirred dead-end ultrafiltration was accurately described. It was clearly demonstrated that the low filtration flux around the isoelectric point is mainly due to the build-up of compact filter cake, which causes a large hydraulic flow resistance. Furthermore, it was shown that the solvent density ρ has a large effect on the dynamically balanced filtration rate in upward ultrafiltration just as it does on the sedimentation coefficient in ultracentrifugation.

Analysis of dead-end ultrafiltration based on ultracentrifugation method

In order to clarify the internal structures of the filter cake formed in protein ultrafiltration, ultracentrifugation experiments were conducted. The local specific flow resistance at the various solution concentrations was determined by the sedimentation velocity technique. The porosity was related to the solute compressive pressure by the high-speed sedimentation equilibrium technique. A method has been developed for evaluating the internal structures in the cake on the basis of a compressible cake resistance model using ultracentrifugation data. The decline in the filtration rate in dead-end ultrafiltration was accounted for by considering the internal structures of the compressible cake. The properties of the cake were determined from ultrafiltration experiments in which a filter was subjected to a sudden reduction in its filtration area. The results accorded well with the calculations. This study revealed that the filtration behavior of ultrafiltration can be accurately described by a compressible cake resistance model.

Studies of the structure of fructose-6-phosphate 2-kinase:fructose-2,6-bisphosphatase.

Some physicochemical properties of a homogeneous preparation of a bifunctional enzyme, fructose-6-phosphate 2-kinase:fructose-2,6-bisphosphatase, were determined. The molecular weight of the enzyme is 101 000 as determined by high-speed sedimentation equilibrium. The molecular weight of dissociated enzyme is 55 000 in 6 M guanidinium chloride by sedimentation equilibrium and in sodium dodecyl sulfate by polyacrylamide gel electrophoresis. A value of 4.7 was observed for the isoelectric point. Tryptic peptide maps and high-performance liquid chromatography of the trypsin-digested enzyme revealed approximately 60 peptides. Amino acid analysis of the enzyme shows that it contains 27 lysine and 36 arginine residues per 55 000 daltons. No free N-terminal amino acid residue was detectable, suggesting that it is blocked. Hydrolysis of the enzyme by carboxypeptidases A and B releases tyrosine followed by histidine and arginine, indicating that the amino acid sequence at the carboxyl terminus is probably -Arg-His-Tyr. Tryptic digestion of [32P]phosphofructose-6-phosphate 2-kinase:fructose-2,6-bisphosphatase yields a 32P-labeled peptide detected by tryptic peptide mapping and high-performance liquid chromatography. Thermolysin digestion of CNBr-cleaved 32P-enzyme also yields a single 32P-peptide. These results indicate that fructose-6-phosphate 2-kinase:fructose-2,6-bisphosphatase is a dimer of 55 000 daltons and the subunits are very similar, if not identical.

Partial amino acid sequence of the wheat germ Ec protein. Comparison with another protein very rich in half-cystine and glycine: wheat germ agglutinin

A wheat germ protein (Ec), the dominant site of cysteine incorporation during early (E) germination of isolated wheat embryos, has been partially sequenced by automated Edman degradation. The sequence of residues 1–59 is not significantly similar to the amino acid sequence known for any other protein, including wheat germ agglutinin which, like Ec, is very rich in half-cystine and glycine. The partial sequence for Ec contains an almost identical pattern of half-cystine residues in segments 6–20 and 35–48, a duplication which includes 10 of the 12 half-cystine residues in the sequence. The partial sequence of Ec may include a large part of the complete sequence, but this remains uncertain because it has not been possible to arrive at a definitve estimate of molecular weight using different physical techniques. Protein Ec can be prepared from a reticulocyte lysate in which cell-free synthesis is programmed by bulk wheat germ mRNA. Determination of the distribution of half-cystine moieties between residues 1 and 20 by Edman degradation of the [35S]cysteine-labeled product of cell-free synthesis shows that it is devoid of an N-terminal extension. Unlike wheat germ agglutinin, Ec does not seem to arise by processing of a conspicuously larger precursor protein. Unlike Ec, another wheat germ protein, Em, the most conspicuous methionine-labeled protein when cell-free protein synthesis is directed by wheat germ mRNA, is refractory to direct sequence analysis by Edman degradation. However, again unlike Ec, uncertainty about the molecular weight of Em, based on its mobility in different sodium dodecyl sulphate – polyacrylamide gel systems, has been resolved by virtue of Em being ideally suited to study by the Yphantis high-speed sedimentation equilibrium method.

Purification and physical-chemical properties of acetyl-CoA: Arylamine N-acetyltransferase from pigeon liver

Acetyl-CoA:arylamine N-acetyltransferase (EC 2.3.1.5) from pigeon liver was purified by protamine sulfate precipitation, ion exchange chromatography on DEAE-A-25 Sephadex, gel filtration on Sephadex G-75, amethopterin-AH-Sepharose 4B affinity chromatography, and finally, gel filtration on Sephadex G-100. The enzyme preparation was homogeneous as judged by ultracentrifugation studies, SDS-polyacrylamide gel electrophoresis and gel filtration. The N-terminal amino acid was detected to be histidine and the complete amino acid composition is reported. The enzyme contains one disulfide bridge and two cysteine residues/mol monomer. The isoelectric point was estimated to be 4.8. The molecular weight was determined to be 32 900 by high-speed sedimentation equilibrium analysis, 33 000 by Sephadex G-100 gel filtration and 31 600 by SDS-disc gel electrophoresis. The sedimentation coefficient from conventional sedimentation velocity runs was 3.1 S observed by ultraviolet optics. ‘Active enzyme centrifugation’ showed a sedimentation constant of 5.0 and 4.8 S for the purified enzyme and crude extract from pigeon liver, respectively, indicating that the enzyme forms a dimer under conditions of catalysis. It could be demonstrated that the inhibitor amethopterin was noncompetitive with respect to the acetyl donor and the acetyl acceptor. Acetyl-CoA:arylamine N-acetyltransferase was examined in different organs of pigeon. The enzyme was not inducible by 1,3-phenylenediamine and hexobarbital in vivo.

The purification, characterization, and activation of phosphoenolpyruvate carboxykinase from chicken liver mitochondria.

The enzyme phosphoenolpyruvate carboxykinase has been purified from chicken liver mitochondria. This purification includes a pseudo-affinity column step utilizing Sepharose 4B-blue dextran which binds the enzyme. The enzyme elutes with ITP to yield protein which is greater than 98% pure. The enzyme has Mr = 75,400 +/- 200 estimated by high speed sedimentation equilibrium and 70,500 +/- 500 estimated by reduced sodium dodecyl sulfate-polyacrylamide gels. The enzyme is abnormally retarded on molecular exclusion resins yielding low apparent molecular weight values. The amino acid analysis indicates that the enzyme has a high proline content and a high tryptophan content and contains 9 mol of cysteine/mol of enzyme. No disulfide bonds were detected. The extinction coefficient (epsilon 1% 280 = 16.5 +/- 0.1) reflects the high tryptophan content. The Svedberg coefficient (s20,w = 4.63 +/- 0.03 S) is consistent with a globular protein of Mr = 70,500-75,400. The activation of the enzyme was investigated by steady state kinetics. The carboxylation reaction has an activation energy of 17.6 kcal/mol. There is no requirement of a monovalent cation for activity. A thiol is necessary for maximal activity, although apparently not to reduce disulfide bonds within the enzyme. Incubation with dithiothreitol stabilizes enzymatic activity but beta-mercaptoethanol facilitates loss of activity. The kinetics of activation by Mn2+ was performed. The Ks value for phosphoenolpyruvate (300 microM) decreases to an apparent Km of 67 microM with increasing concentrations of Mn2+. The concentration of Mn2+ does not affect the interaction of HCO-3 with the enzyme, however. Analysis of data in terms of free IDP indicates that increasing Mn2+ decreases the Km of IDP but analysis as MnIDP indicates the Km,app of MnIDP is independent of the Mn2+ concentration. The enzyme interacts with Mn2+ with a KA = 67 microM and the Km,app decreases to a value of 8 microM with saturating substrates. The substrate analogue (Z)-3-fluorophosphoenolpyruvate is a good substrate for the reaction (Km = 30 microM) with 27% Vmax compared to P-enolpyruvate (Km = 180 microM). Except for 3-bromophosphoenolpyruvate, other analogues have shown weak competitive or noncompetitive inhibition. Potential analogues of oxalacetate (succinate, citrate, isocitrate, malate, and alpha-ketoglutarate) all elicit weak (greater than 15 mM) inhibition.

Thermodynamics of concanavalin A dimer-tetramer self-association: sedimentation equilibrium studies.

The effects of temperature and pH on the reversible dimer-tetramer association of concanavalin A were studied by the high-speed sedimentation equilibrium technique. Both commercial and highly purified preparations of concanavalin A were used. Equilibrium constants were analyzed as a Wyman linked function by using truncated van't Hoff temperature dependence. In the concentration range 0.1-3.0 mg/mL, from 5 to 35 degrees C and between pH 5.5 and pH 7.5 at 0.5 M ionic strength, only dimer and tetramer species were present in both preparations. For purified concanavalin A, association constants ranged from 1.5 X 10(3) to 8.0 X 10(7) M-1. Constants for our commercial preparation were approximately 10-fold lower due to the decrease competency of some subunits to self-associate. From the fit of the Wyman model to the experimental data, delta G degree, delta H degree, delta S degree, and delta Cp degree were calculated for the association and association-linked ionization reactions. From the values of the ionization thermodynamic parameters, the association is governed by the ionization of a histidine side chain on each subunit, either histidine-51 or histidine-121. The association is characterized by large entropy (66.3 cal.mol-1.deg-1 at 25 degrees C) and heat capacity (-821 cal.mol-1.deg-1) changes in accordance with the large hydrophobic association surface observed in crystallographic studies [Reeke, G. N., Jr., Becker, J. W., & Edelman, G. M. (1975) J. Biol. Chem. 250, 1525-1547]. In addition, there is a large enthalpy change (10.4 kcal.mol-1 at 25 degrees C). We propose a model for the interaction based on a more detailed thermodynamic description than was obtained in an earlier, incomplete study [Huet, M., & Clavarie, J. M. (1978) Biochemistry 17, 236-241].

Effects of saccharide and salt binding on dimer-tetramer equilibrium of concanavalin A.

The effect of the binding of saccharide ligands on the reversible dimer-tetramer equilibrium on concanavalin A was studied by the high-speed sedimentation equilibrium technique. Both commercial and highly purified fragment-free concanavalin A preparations were used. In the case of the fragment-free preparation, there was no effect of the binding of alpha-methyl mannoside or alpha-methyl glucoside at 35 degrees C and at a variety of conditions of pH and ionic strength. This implies no difference in ligand binding activity between dimeric and tetrameric Con A, in contrast to an earlier report [McKenzie, G. H., & Sawyer, W. H. (1973) J. Biol. Chem. 248, 549-556]. There was a profound effect in the case of the commercial preparation. Dimers that contain hydrolyzed subunits appear to be incompetent to self-associate in the presence of alpha-methyl mannoside or alpha-methyl glycoside, while alpha-methyl galactoside, which does not bind to Con A, had no effect. The effects of very high concentrations of CaCl2 (to 2.5 m) and NaCl (60 6.2 m) were also studied. The data were analyzed by an integrated form of the Tanford extension [Tanford, C. (1969) J. Mol. Biol. 39, 539-544] of the Wyman linked function theory, which includes preferential interactions with salt and water. The integrated form allows preferential interactions to be described as the sum of salt binding and water binding. The data were well described by salt binding alone; it was unnecessary to invoke any water binding effect. The CaCl2 data did indicate that one calcium per subunit of the dimer binds to a site that is buried in the tetramer. This suggests a site on the dimmer-dimer interface which is consistent with Reeke's identification of the protomers composing the solution dimer [Reeke, G. N., Jr., Becker, J. W., & Edelman, G. M. (1975) J. Biol. Chem. 250, 1525-1547].

Isolation from erythrocytes of a green hemoprotein with ferroactivator activity.

A protein that exhibits ferroactivator activity with purified rat liver P-enolpyruvate carboxykinase was purified to apparent homogeneity from bovine erythrocytes. The homogeneous protein has a molecular weight of 82,000 by high speed sedimentation equilibrium and 90,000 by gel exclusion chromatography. A subunit molecular weight of 22,500 was obtained by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Its isoelectric focusing indicated that the ferroactivator protein has an isoelectric point at pH 5.7. The reduced pyridine hemochrome of the denatured protein shows absorption maxima at 419, 527, and 557 nm which is characteristic of protoheme IX. The native protein is green in color with absorption maxima at 407, 507, and 543 nm. The amino acid composition and immunological studies show that bovine erythrocyte protein is structurally similar to the P-enolpyruvate carboxykinase ferroactivator protein isolated from rat liver. Its function in the erythrocyte is unknown.

Large-scale purification and some properties of malate synthase from baker's yeast.

1. Malate synthase from baker's yeast (5 kg) was purified 400--500-fold to homogeneity. About 50--200 mg homogeneous enzyme were obtained within a week in a yield of 30% with reference to the total activity in cell-free crude extracts. The enzyme, pI = 7.5, was pure as judged from ultracentrifugal and gel electrophoretic studies. 2. Sedimentation and diffusion coefficients were determined: S 0 20,w = 8.26 +/- 0.05 S, D 0 20,w = 4.5 +/- 0.1 X 10(-7) cm2 s-1. The molecular weight of the synthase was found to be 175 000 +/- 10 000 and 180 000 +/- 10 000 by sedimentation/diffusion and by high-speed sedimentation equilibrium respectively. It was concluded from these and other results that malate synthase has a molecular mass of 180 000 +/- 10 000. 3. The synthase on sodium dodecylsulfate gel electrophoresis was dissociated to yield a single specimen of Mr about 66 000. The result indicates a composition of the native enzyme from three subunits of identical or nearly identical mass. 4. The binding of acetyl-coenzyme A to the synthase is independent of Mg2+ but that of glyoxylate is strictly dependent on the presence of Mg2+. Kinetic studies indicate that the malate synthase reaction follows a sequential random mechanism. 5. The intermolecular isotopic effect, kH:k2H = 1.4, was determined with acetyl-coenzyme A and [2H3]acetyl-coenzyme A under several different experimental conditions and was shown to reflect different maximal velocities of the two substrates. An enzymic procedure for the preparation of doubly labelled [3H, 14C]acetyl-coenzyme A is also presented.

Thermodynamic studies of concanavalin a dimer-tetramer equilibria

We have studied the reversible dimer-tetramer association equilibria of the jack bean lectin concanavalin A. Equilibrium constants were measured by the high speed sedimentation equilibrium technique as a function of pH, temperature, and CaCl/sub 2/ concentration. Both commercial and purified preparations of concanavalin A were studied.

Salt-dependent interconversion of inner histone oligomers

The inner histone complex, extracted from chicken erythrocyte chromatin in 2 M NaCL AT pH 7.4, has been characterized by sedimentation equilibrium and sedimentation velocity. High speed sedimentation equilibrium studies indicate that in 2 M NaCl the inner histones are a weakly associating system with contributions from species ranging in molecular weight from dimer to octamer. The appearance of a single boundary (3.8S at 2 M NaCl) in sedimentation velocity studies conducted over a wide range of protein concentrations and ionic conditions indicates that the various histone oligomers present are in rapid equilibrium with one another. At higher salts the equilibrium is shifted to favor higher molecular weight species; in 4 M NaCl essentially all of the histone is octameric at protein concentrations above 0.2 mg/ml. The facile interconversion of histone oligomers suggests that small alterations in histone-histone interactions may be responsible for changes in nucleosome conformations during various biological processes.

Canine cardiac myosin with special referrence to pressure overload cardiac hypertrophy. I. Subunit composition.

In studies of myosin from left and right ventricles of normal hearts and hypertrophic hearts at 5 weeks and 13 weeks after aortic banding, polyacrylamide gel electrophoresis shows intermediate molecular weight components which derive from heavy chains fragmented in the presence of dodecyl sulfate. The proportion of degraded heavy chains is greater in myosin from hypertrophic hearts than normal hearts, with comparable degradation in left and right ventricle myosin. The observed fragmentation of myosin results from proteolysis due to contaminant proteases or a thermally activated, heat-stable nonenzymatic process, or both. The susceptibility of heavy chains to crude myofibrillar proteases differs in normal and hypertrophic cardiac myosin; however, the kinetics of tryptic digestion are identical for both myosins. With precautions to minimize proteolytic artifacts on dodecyl sulfate-polyacrylamide gel electrophoresis, preparations of myosin from left and right ventricles of normal and hypertrophic hearts exhibit comparable subunit composition, with approximately molar ratios of heavy chains, light chain L1, and light chain L2. Comparable stoichiometry for the light chain fraction is determined by high speed sedimentation equilibrium at pH 11 and direct fractionation of the different cardiac myosins. We do not confirm reports (e.g. Wikman-Coffelt, J., Fenner, C., Smith, A., and Mason, D. T. (1975) J. Biol. Chem. 250, 1257-1262) of different proportions of light chains in left and right ventricle myosin of normal and hypertrophic canine hearts. The light chains display microheterogeneity, with L1 generating two isoelectric variants and L2 generating two major and two minor variants, but identical mobilities and isoelectric values are obtained in the different myosin preparations.

Characterization of group A streptococcal T-12 protein purified by ion-exchange column chromatography

The aim of the present study was to describe the physicochemical characteristics of streptococcal T antigen. T protein isolated from Streptococcus pyogenes type 12 (R53/1077, Colindale) and purified by ion-exchange column chromatography resulted in a preparation that was homogeneous when tested electrophoretically (in two systems, in presence and in absence of sodium dodecyl sulfate) and by gel filtration on Sephadex G-100. The purified T antigen was resistant to enzymatic degradation by trypsin and pepsin. It formed a single precipitin line with standard T-12 antiserum and was not contaminated with group A carbohydrate and M protein. The molecular weight of protein T, determined by means of polyacrylamide gel electrophoresis and calculated from its amino acid composition, was about 39,000. The molecular weight of this protein, determined by means of high-speed sedimentation equilibrium, ranged between 80,000 and 120,000. Glutamic and asparatic acids, lysine, alanine, and leucine were the predominant amino acids.

Subunit association and heterogeneity of Limulus polyphemus hemocyanin.

The molecular weights of the 6S, 24S, 36S, and 60S components of Limulus polyphemus hemocyanin were determined by high speed sedimentation equilibrium to be 69 400, 856 000, 1 690 000, and 3 160 000. The behavior of this hemocyanin appears to be similar to that of other arthropod hemocyanins where the first aggregation step is the formation of a hexamer of the 6S monomer. Here the larger aggregated states (24S, 36S, and 60S) are successive dimers of an unobserved hexamer (16S). The 24S-36S-60S association was found to be heterogeneous, suggesting that 24S components of different composition may be present.

Tobacco mosaic virus protein: sedimentation equilibrium studies of the initial stages of polymerization.

The lowest stages of polymerization of tobacco mosaic virus protein were studied by means of high-speed sedimentation equilibrium experiments. Several distinct modes of polymerization were found. At pH 7.1 the expected monomer-trimer-higher polymer equilibrium was observed--very little dimer was detected at this pH. At pH 7.5, however, a strong dimerization was observed--neither monomer nor trimer was detected at this pH. An octamer appeared to be the only species present other than the dimer. When 0.01 M beta-mercaptoethanol was added to the solvent pH 7.5, the dimer was dissociated, resulting in a monomer-trimer association. The dimerization may be the basis for the larger "doubled" polymers formed by the protein at alkaline pH, while the octamer may correspond to the 8S peak frequently observed in sedimentation velocity experiments at alkaline pH. On the other hand, the monomer-trimer-higher polymer equilibrium may correspond to the single helix formed by the protein at slightly acid pH and to the combination of 4S and 20S peaks seen in sedimentation velocity experiments at slightly acid pH.

Effect of purification procedures on the self-association of myosin at high ionic strength

The effects of purification procedures on the self-association of myosin at high ionic strength have been investigated by high-speed sedimentation equilibrium. The method of purification profoundly affects the self-association characteristics of the myosin molecule. Myosin purified by chromatography on Sephadex G-200 (S-myosin) behaves (in the limit) as a single nonideal thermodynamic component with little (if any) reversible dimerization ( K 21 = 1.05 dl/g) and with a second viral coefficient, B 1 = 0.064 dl/g, compatible with excluded volume effects alone. Conversely, myosin which had been subjected to (NH 4) 2SO 4 precipitation followed by chromatography on DEAE-Sephadex (DEAE-myosin) dimerizes reversibly to a significant extent judged by its apparent association constant, K 21 ≅ 9 dl/g. In addition, higher N-mers appear to partake in the equilibria. The nonideality of this system, B 1 = 2.2 ± 0.1 dl/g, is also greater than that of S-myosin. Two possible explanations to account for these findings are offered: (1) Myosin may undergo a change in conformation (denaturation?) during the preparation steps which lead to DEAE-myosin; (2) another component (C-protein?) which would modulate the self-association properties of myosin may be involved.