Increase In Sedimentation Coefficient Research Articles

Full hydrodynamic reversibility of the weak dimerization of vancomycin and elucidation of its interaction with VanS monomers at clinical concentration

The reversibility and strength of the previously established dimerization of the important glycopeptide antibiotic vancomycin in four different aqueous solvents (including a medically-used formulation) have been studied using short-column sedimentation equilibrium in the analytical ultracentrifuge and model-independent SEDFIT-MSTAR analysis across a range of loading concentrations. The change in the weight average molar mass Mw with loading concentration was consistent with a monomer-dimer equilibrium. Overlap of data sets of point weight average molar masses Mw(r) versus local concentration c(r) for different loading concentrations demonstrated a completely reversible equilibrium process. At the clinical infusion concentration of 5 mg.mL−1 all glycopeptide is dimerized whilst at 19 µg.mL−1 (a clinical target trough serum concentration), vancomycin was mainly monomeric (<20% dimerized). Analysis of the variation of Mw with loading concentration revealed dissociation constants in the range 25-75 μM, commensurate with a relatively weak association. The effect of two-fold vancomycin (19 µg.mL−1) appears to have no effect on the monomeric enterococcal VanS kinase involved in glycopeptide resistance regulation. Therefore, the 30% increase in sedimentation coefficient of VanS on adding vancomycin observed previously is more likely to be due to a ligand-induced conformational change of VanS to a more compact form rather than a ligand-induced dimerization.

The cyanobacterial repressor SmtB is predominantly a dimer and binds two Zn2+ ions per subunit.

The Synechococcus PCC7942 metallothionein repressor gene smtB has been cloned into a high expression vector and the protein purified to near homogeneity (>/=98%). Analytical ultracentrifugation studies demonstrate that the protein is predominantly dimeric in 0.1 M NaCl, pH 7.4, and 22 degrees C, exhibiting a monomer-dimer-tetramer equilibrium. The monomer-dimer (Ka(1,2)) and the dimer-tetramer (Ka(2,4)) association constants are 3.24 x 10(5) and 9.90 x 10(2) M-1, respectively. The repressor binds two Zn2+ ions per subunit with an overall Kd of 3.49 x 10(-6) M. In the presence of Zn2+, Ka(1, 2) increases by 2 orders of magnitude to 1.25 x 10(7) M-1 and the apparent weight-averaged sedimentation coefficient increases from 2. 00 to 2.22 S. The fact that the increase in sedimentation coefficient is greater than that predicted by increased dimerization is interpreted as caused by compaction of the structure in the presence of metal ions. At pH 6.0, 0.1 M NaCl, and 22 degrees C, the protein exhibits only a monomer-dimer equilibrium, with Ka(1,2) = 1.52 x 10(7) M-1 which is almost identical to that seen upon binding Zn2+ at pH 7.4. The compaction and conformational change in SmtB caused by Zn2+ is consistent with a role for this altered quaternary state in derepression of smtA in Synechococcus challenged with heavy metal ions.

Thermodynamic nonideality in enzyme catalysis. Effect of albumin on the reduction of pyruvate by lactate dehydrogenase.

The enhanced catalytic reduction of pyruvate by rabbit muscle lactate dehydrogenase that results from the addition of serum albumin [Nichol, L. W., Sculley, M. J., Ward, L. D. & Winzor, D. J. (1983) Arch. Biochem. Biophys. 222, 574-581] is shown to emanate solely from an increase in maximal velocity, there being no discernible effect of this inert space-filling macromolecular solute on the Michaelis constant for either pyruvate or NADH. As part of the search for a mechanistic explanation of this kinetic phenomenon, the space-filling effects of albumin have been used to eliminate the possibility that the increase in sedimentation coefficient of lactate dehydrogenase effected by inclusion of oxamate with enzyme-NADH complex reflects preferential binding of this pyruvate analog to a more compact isomeric state of the binary complex. The enzyme kinetic results are therefore considered in terms of a reaction scheme entailing gross conformational changes during the formation of ternary enzyme-NADH-pyruvate complex and its isomerization to an activated transition state. The experimentally observed insensitivity of the Michaelis constant for pyruvate to albumin concentration is in keeping with theoretical prediction, but incorporation of the measured extent of maximal velocity enhancement into the kinetic model leads to a predicted volume for the fully saturated transition-state complex that is far too small to be experimentally feasible. A more complex mechanistic model involving additional isomerizations of enzyme-substrate species is thus required to achieve quantitative description of the albumin effect solely in terms of thermodynamic nonideality.

Tamoxifen-estrogen receptor complex: Activation and immunorecognition by a monoclonal antibody

1.1. Large chicken oviducts containing an optimal amount of the cytoplasmic estrogen receptor can be obtained by the application and short-term withdrawal of diethylstilbestrol implants to chickens.2.2. The cytoplasmic estrogen-receptor complex from chicken oviduct is able to bind to DNA. The efficiency of the binding (maximal 60%) is dependent on the temperature and the presence of the hormone.3.3. The strogen-receptor complex eluted from the DNA-cellulose by high salt is different from that of untreated cytosol. The observed increase in sedimentation coefficient, molecular weight and assymetry of the receptor molecule resembles the ‘transformation’ of the strogen-receptor complex from rat uterus.4.4. Using DNA-cellulose and agarose gel chromatography, the cytoplasmic estrogen-receptor complex from chicken oviduct cna be purified 4000-fold.

Forskolin-induced change of the size of adenylate cyclase.

Forskolin, a potent activator of cyclic AMP generating systems, has been proposed to act directly on the catalytic unit of adenylate cyclase. Nevertheless, some arguments indicate a possible role of the guanosine triphosphate-binding regulatory protein in forskolin action on adenylate cyclase. In this study, we have observed an increase in the apparent sedimentation coefficient of solubilized adenylate cyclase, elicited by forskolin, both in rat liver (from 6.4 +/- 0.1 to 7.2 +/- 0.1 S) and rat striatum (from 6.7 +/- 0.1 to 7.6 +/- 0.1 S). On both systems, a similar increase in the sedimentation coefficient was observed after preactivation of the enzyme with guanosine 5'-(beta, gamma-imido)triphosphate (Gpp(NH)p). In contrast to the Gpp(NH)p effect, the forskolin action was found to be reversible. Simultaneous pretreatments of adenylate cyclase with forskolin and Gpp(NH)p did not induce additive increases of the apparent sedimentation coefficient of adenylate cyclase. The modification of the size of solubilized adenylate cyclase was corroborated by gel filtration studies. In rat liver membranes, the Stokes radius of the solubilized enzyme increased from 59 +/- 1 A for basal state to 65 +/- 1 A for forskolin preactivated state. A possible explanation of our findings is that forskolin may stabilize the complex between the GTP-binding regulatory protein and the catalytic unit of adenylate cyclase in a reversible manner.

Estrogen receptor in chicken oviduct: Receptor dissociation kinetics and transformation

Cytosolic and nuclear estrogen receptor forms of chicken oviduct have been studied by (1) measuring hormone dissociation kinetics and by (2) sucrose density gradient analysis on high salt gradients. Estradiol dissociates from the receptor in chicken oviduct cytosol at 22°C following a two-phase exponential process. The fraction of receptor with a fast dissociation rate ( k = 120 × 10 −3min −1) decreases as a function of the pre-incubation at 22°C: after prolonged pre-incubation only the slowly dissociating ( k = 12.3 × 10 −3min −1) form remains. Dissociation of moxestrol, a synthetic estrogen with a higher affinity, from the cytosol receptor at 30°C is similar, showing a transition of a fast dissociating form ( k = 120 × 10 −3min −1) to a slowly dissociating form ( k = 7.6 × 10 −3min −1) as a result of pre-incubation at 30°C. A concomitant temperature-dependent shift of the estrogen receptor from a 4.8 S to a 6.1 S form was observed with moxestrol but not with estradiol as a ligand. Sodium molybdate (20 mM) and NaSCN (400 mM) inhibit the temperature-dependent increase in sedimentation coefficient, but molybdate allows the formation of a receptor form which shows intermediary dissociation kinetics. Estrogen receptor, precipitated with ammonium sulfate (0–35%) shows monophasic dissociation kinetics of estradiol ( k =39.5 × 10 −3min −1) and for moxestrol ( k = 10.8 × 10 −3min −1), suggesting full receptor activation only with moxestrol as a ligand. Moxestrol-receptor complexes obtained by ammonium sulfate precipitation sediment at 0°C at 4.8 S. Only after subsequent incubation at 30°C a shift from 4.8 S to 5.9 S is observed, suggesting that the formation of the slowly dissociating form of the receptor may precede the formation of a stable transformed receptor complex. The nuclear estrogen receptor with estradiol as a ligand shows biphasic dissociation kinetics at 22°C ( k = 10 × 10 −3min −1; k = 14.0 × 10 −3min −1). The ratio of both components (1:1) does not change after preincubation of the nuclear receptor extract at 22°C. Moxestrol dissociates from the nuclear receptor at 30°C monophasically with a slow rate ( k = 6.1 × 10 −3min −1), suggesting that it is extracted as an activated hormone-receptor complex. Our data show that in vitro the cytosolic estrogen receptor can undergo a temperature-dependent transition from a low to a high affinity form. The in vitro transformation to a form with a higher sedimentation coefficient appears to be dependent on the type of ligand bound to the receptor. The receptor extracted from the nuclei is predominantly present in the higher affinity state.

Evidence for similar conformational changes in alpha 2-macroglobulin on reaction with primary amines or proteolytic enzymes.

Reactions of alpha(2)-macroglobulin (alpha(2)M) with primary amines (ammonium chloride, methylammonium chloride and ethylammonium chloride) or proteolytic enzymes (trypsin, chymotrypsin and thrombin) resulted in changes of the absorption, fluorescence and circular-dichroism spectra and of the sedimentation coefficient of the inhibitor. All physico-chemical changes caused by the inactivation of alpha(2)M by the amines were identical with, or highly similar to, those induced by the formation of the enzyme-inhibitor complexes. This suggests that similar conformational changes of the inhibitor occur in the two types of reactions. The frictional ratio, calculated from the increase in sedimentation coefficient, decreased from 1.67 for untreated alpha(2)M to 1.57 for the amine- or proteinase-treated inhibitor. This change is due to a decrease in either asymmetry or hydration of the protein, resulting in a slightly smaller hydrodynamic volume. The circular-dichroism analyses indicated that the reaction of alpha(2)M with either amines or proteinases is accompanied by a loss of the small amount (about 5%) of alpha-helix of the untreated protein. The changes of u.v. absorption and fluorescence suggested that about one out of the eight to ten tryptophan residues of each alpha(2)M subunit is buried as a result of the conformational change. All spectroscopic and hydrodynamic changes that were observed are compatible with a spatial rearrangement of the subunits of alpha(2)M, as implicated by the ;trap' hypothesis for the mechanism of inhibition of proteinases. However, a conformational change involving a decrease in the hydrodynamic volume of each subunit cannot be excluded.

Repair of thermal damage to the Escherichia coli nucleoid.

The folded chromosome or nucleoid of Escherichia coli was analyzed by low-speed sedimentation in neutral sucrose gradients after heat treatment (30 min at 50 degrees C) and subsequent incubation of cells at 37 degrees C for various times. Heat treatment resulted in in vivo association of the nucleoids with cellular protein and in an increase in sedimentation coefficient. During incubation at 37 degrees C, a fraction of the nucleoids, from heated cells, because dissociated from cellular protein and regained their characteristic sedimentation coefficients. The percentage of nucleoids which returned to their control sedimentation position in the sucrose gradients corresponded to the percentage of cells able to repair thermal damage as assayed by enumeration on agar plates.

A circular dichroism study on the structure of DNA and the nucleosomal core particle using Hg(II) and Ag(I)

CD studies of known sequence DNA-like polynucleotide-Hg 2+ complexes have established a cross-strand binding of Hg 2+ to the two thymines of the base-paired first-neighbor unit TpA, as the type I binding mode of poly[d(A-T) · d(A-T)] and other polynucleotides with high TpA incidence. The amino group of adenine is the most probable Hg 2+ site for poly(A) and for the binding mode II of poly[d(A-T) · d(A-T)]. The binding of Hg 2+ to the natural DNAs shows two binding modes with two different degrees of CD perturbation. The two proposed binding modes are substantiated by Hg 2+ binding studies with the DNA-poly( l-lysine) complexes. The increase of sedimentation coefficient of the nucleosomal core particle-heavy metal ion complexes can be accounted for by the binding of heavy metal ions. From the CD experiments with Hg 2+ and Ag +, it is estimated that 80 ± 10% of the DNA in the nucleosomal core particle is involved in the wrapping around the histone core. An out-of-plane transition of the DNA-Ag + binding mode II is proposed as the cause for the large CD perturbation at 270 nm, but the origin of optical activity of the DNA-Hg 2+ complex is still unknown.

DNA-binding ability and partial purification of the cytoplasmic estrogen receptor from chicken oviduct

1. 1. Large chicken oviducts containing an optimal amount of the cytoplasmic estrogen receptor can be obtained by the application and short-term withdrawal of diethylstilbestrol implants to chickens. 2. 2. The cytoplasmic estrogen-receptor complex from chicken oviduct is able to bind to DNA. The efficiency of the binding (maximal 60%) is dependent on the temperature and the presence of the hormone. 3. 3. The strogen-receptor complex eluted from the DNA-cellulose by high salt is different from that of untreated cytosol. The observed increase in sedimentation coefficient, molecular weight and assymetry of the receptor molecule resembles the ‘transformation’ of the strogen-receptor complex from rat uterus. 4. 4. Using DNA-cellulose and agarose gel chromatography, the cytoplasmic estrogen-receptor complex from chicken oviduct cna be purified 4000-fold.

Rearrangements in the Course of Ribonuclease Hydrolysis of Pre‐messenger Ribonucleoproteins A Warning

A study of the action of ribonuclease on 30--50-S monoparticles prepared from pre-messenger ribonucleoprotein (pre-mRNA . protein) was started in order to elucidate the structure of monoparticles. A ribonucleoprotein complex containing mostly 30000--38000-Mr proteins of pI 7--9 (alpha class) persisted under conditions where other proteins (23000--110000 Mr, pI 5--8.5, beta class) were relased. An unexpected increase of sedimentation coefficient accompanied the formation of the ribonucleoprotein complex. The extent of increase varied with the initial size of the monoparticles, reaching 45% for 30-S monoparticles. The ribonucleoprotein complexes designated here as 40--45-S alpha-ribonucleoproteins were more homogeneous in size than the original monoparticles. Electron microscopic examination showed that the sedimentation shift corresponded to an increase of the actual size of the particles, not to flattening or change of shape. Therefore, the 40--45-S alpha-ribonucleoprotein is not a pre-existing unit of pre-mRNA . protein but arises from specific rearrangements probably between small alpha ribonucleoproteins formed by fragmentation of monoparticles. In addition to the 40--45-S alpha-ribonucleoproteins, large protein aggregates corresponding to 15% of the monoparticle proteins were formed upon ribonuclease hydrolysis. Their major proteins were neutral, suggesting that the aggregates might be precipitates of proteins at pH close to the pI. Ribonuclease being a widespread cellular enzyme, partial rearrangements may occur during preparation and handling of pre-mRNA . protein. It is particularly crucial to remark that the 40--45-S alpha-ribonucleoprotein which does not pre-exist might be mistaken for a pre-mRNA . protein unit.

Structural transition in chromatin induced by ions in solution.

Structural transition in chromatin was measured as a function of counter ions in solution (NaCl or MgCl(2)) and of histones bound on the DNA. The addition of counter ions to aqueous solutions of chromatin, partially dehistonized chromatin, and DNA caused a drastic reduction in viscosity and a significant increase in sedimentation coefficient. Transitions occurred primarily at about 2 x 10(-3) M NaCl and 1 x 10(-5) M MgCl(2) and are interpreted as a change in structure of chromatin induced by tight binding of cations (Na(+) or Mg(++)) to DNA, either free or bound by histones, and is an intrinsic property of DNA rather than of the type of histone bound. At a given ionic condition, removal of histone H1 from chromatin had only a minor effect on the hydrodynamic properties of chromatin while removal of other histones caused a drastic change in these properties. An increase in the sedimentation coefficient of DNA was observed also for protamine. DNA complexes wherein the bound protein contains only unordered coil rather than the alpha-helices found in histones.

Knotted single-stranded DNA rings: A novel topological isomer of circular single-stranded DNA formed by treatment with Escherichia coli ω protein

Treatment of single-stranded circular phage fd DNA with Escherichia coli ω protein yields a new species which sediments 1.2 to 1.5 times faster than the untreated DNA in an alkaline medium. The infectivity of this species in spheroplast assays, after purification of the DNA by zone sedimentation in an alkaline sucrose gradient, is only slightly lower than that of untreated fd DNA. The formation of this species requires Mg(II) and is strongly dependent on salt concentration and temperature. At 37 °C, over 85% of the input DNA can be converted to this form when incubation is carried out in media containing 0.15 to 0.25 m-salt. The yield decreases with increasing temperature or decreasing salt concentration. The increase in sedimentation coefficient of fd DNA in an alkaline medium following treatment with ω is not due to protein binding, as no change was observed upon treatment of the product with phenol or Pronase. Furthermore, neither the buoyant density of this new species in neutral CsCl nor its sedimentation coefficient in a neutral medium is significantly different from the corresponding properties of untreated fd DNA. Examination by electron microscopy shows that the new form has the appearance of a knotted ring of about the same contour length as an untreated monomeric single-stranded fd DNA. The new form can be converted to full-length linear fd DNA by treatment with pancreatic DNAase I. The rate of conversion is approximately the same as that of untreated circular fd DNA to the linear form. These results show that the new form of fd DNA is a novel topological isomer: a knotted single-stranded DNA ring. It is also found that further treatment of the knotted DNA rings with ω at low ionic strength can reverse the reaction, i.e. the knotted DNA rings can be converted back to simple DNA rings indistinguishable from fd DNA from the phage. At intermediate ionic strength the two forms are interconvertible and form an equilibrium mixture. Results similar to those obtained for fd DNA have also been observed for single-stranded circular φX174 DNA. A mechanism based on the known activity of ω protein on double-stranded DNA, the secondary structure of a single-stranded circular DNA, and the experimental results described here is proposed.

Amylose-iodine complex. I. Sedimentation behavior.

Sedimentation measurements are reported on solutions of blue amylose-iodine complexes in the range of 0.001 to 0.007% amylose. Amylose fractions B and F2, of weight average molecular weight average molecular weight 4.0 X 10(5) and 3.4 X 10(4), respectively, were used in this study. Iodine complexes of these fractions formed polydisperse solutions of limited solubility and stability. Sedimentation coefficients increased as a function of potassium iodide concentration. Values for fraction B complexes varied from (16.3 +/- 1.0) X 10(-13) at 1.2 X 10(-3) M KI to (57.2 +/- 7.5) X 10(-13) at 8.3 X 10(-3) M KI; values for fraction F2 complexes varied from (10.0 +/- 1.2) X 10(-13) at 1.2 X 10(-13) M KI to (24.8 +/- 3.9) X 10(-13) at 9.5 X 10(-3) M KI. At constant potassium iodide concentration, sedimentation coefficients, within our experimental error of 10 to 15% standard deviation, are independent of amylose concentration. Time dependence of sedimentation coefficient values was observed for solutions either saturated or unsaturated with respect to the iodine-binding capacity of amylose. For iodine-saturated complex solutions, sedimentation coefficients extrapolated to zero potassium iodide concentration were two to three times greater than for the parent amylose. Measurements are evaluated in terms of possible polyelectrolytic charge effects and aggregation. Under conditions used in these experiments, aggregation of amylose-iodide complexes appears to be the mechanism responsible for the large increase in sedimentation coefficients.

On the interaction of alpha-lactalbumin and galactosyltransferase during lactose synthesis

The regulatory effect of alpha-lactalbumin in the lactose synthase system has been ascribed to its reversible association with a complex of galactosyltransferase with Mn2+ and UDP-galactose, prior to the binding of monosaccharides; the resulting complex has a higher affinity for various monosaccharides. Two steps in the postulated catalytic cycle have been investigated; UDP-galactose binding to enzyme-Mn2+ by equilibrium dialysis and alpha-lactalbumin binding to enzyme-Mn2+-UDP-galactose by sedimentation velocity and kinetics. There is a single binding site for UDP-galactose on the enzyme-Mn2+ complex, and the dissociation constant for UDP-galactose from enzyme-Mn2+-UDP-galactose was found to be 72 muM at 37 degrees. The formation of a complex between galactosyltransferase and alpha-lactalbumin in the presence of Mn2+ and UDP-galactose was observed as an increase in sedimentation coefficient of enzyme activity So20,w from 3.25 +/- 0.03 in the absence of alpha-lactalbumin to 4.22 +/- 0.03 at saturating concentrations of alpha-lactalbumin, a value closely similar to that of a cross-linked 1:1 complex of the proteins under the same conditions (4.35 +/- 0.03). No interaction was observed in the absence of substrates or with UDP-galactose and EDTA. From the ultracentrifuge data and steady state kinetics, dissociation constants for alpha-lactalbumin from the enzyme-Mn2+-UDP-galactose-alpha-lactalbumin complex were determined at several temperatures and salt concentrations. These showed good internal agreement. The free energy change delta G degrees for the association of the two proteins is calculated, and the results are discussed in relation to the nature of the interaction.

Physico-chemical properties of the copper(II)-poly-4-vinylpyridine complexes

Water-soluble Cu(II) complexes of poly-4-vinylpyridine, partially quaternized by methyl bromide or dimethylsulphate (PP, Mt) and of the analogue (4-ethylpyridine) were studied by physicochemical technique such as visible spectrophotometry, viscosity, speed sedimentation and EPR spectroscopy. Peculiarities of the complex formation reaction were observed for the polymer compared to the analogue. The predominant formation of tetrapyridinate-Cu(II) species [(CuL 4)] 24 was found to take place in aqueous solution for PP, Mt with degree of quaternization (β) 20,24,37,49,57,65 per cent over a wide range of P YJ Cu(II) molar ratios. In addition, not all the free polymer pyridine residues are capable of forming [Cu L4 ] 2 complexes even though there is a large excess of Cu(II) ions. The maximum value of the pyridine fraction forming [Cu L 4 ] 21 in PP YMt-20, 24, 37, 49 (obtained by spectroscopic titration) was found to be 45–50 per cent. Addition of Cu(NO 3) 2 to the aqueous polymer solution causes strong reduction of specific viscosity and increase of sedimentation coefficient from - 1 S to - 6 S. These data lend to the suggestion of the existence of macromolecular associates bound by Cu(II) ions and approximate estimation of their average molecular weight. It was shown also that Bjerrum's procedure cannot be used to account for the polymer stability constants. The differences between complex formation for polymer pyridine and monomer pyridine seem to be due to the high local pyridine concentration in the coil of the macromolecule and the hydration effects of the polymer chain on the characteristics of the pyridine residues.

Physical and chemical changes in alpha-crystallin during maturation of lens fibers

A mechanical separation of calf lens into three fractions composed of outer cortex, inner cortex and nucleus provided material representing three phases of fiber maturation. Separation of the lenticular proteins in each fraction was achieved by gel filtration on agarose 15 m. The amount of high mol. wt. alpha-crystallin aggregates was found to increase appreciably as the fibers age. The amount of low mol. wt. alpha-crystallin, however, decreases while the average size gradually increases as the fibers are displaced toward the center of the lens. An increase in sedimentation coefficient corresponding to an increase of 50% in mol. wt. was found when the low mol. wt. alpha-crystallin from the outer cortex was compared to that from the nuclear region. No detectable change in the amino acid or the subunit composition accompanied this increase in size. The circular dichroism (CD) of these crystallins also revealed no conformation changes of the polypeptide backbone. Unique to the CD spectrum for the nuclear alpha-crystallin are minima at 235, 214 and 206 nm. This observation was corroborated by far u.v. spectroscopy to 195 nm. The loss of these CD minima as well as changes in the u.v. profile that can be effected by 7 m-urea suggests the presence of chromophore(s) attached non-covalently to the nuclear protein. One or more other chromophore(s) common to all low mol. wt. alpha-crystallins were revealed by a 40% reduction in the extinction at 195 nm after urea treatment of the cortical alpha-crystallins with no associated change in the CD spectrum. The 20% reduction in extinction for the nuclear protein suggests the presence of similar chromophore(s). Deaggregation followed by reaggregation not only markedly diminished the size of the low mol. wt. alpha-crystallins but also drastically reduced the disparities in size of this protein among the three fiber fractions. These results suggest that chromophore(s) removed by urea treatment may play a decisive role in determining the size of the alpha-crystallin marcromolecule during the maturation of the lens fibers.

Effect of 1-anilino-8-naphthalenesulfonate on properties and associations of .ALPHA.3-,.BETA.- and .KAPPA.-caseins.

It was indicated from fluorescence spectra and fluorescence titration that a hydrophobic probe, 1-anilino-8-naphthalenesulfonate (ANS), binds to casein components (αs-, β- and κ-caseins). Fluorescence intensity and affinity of ANS-κ-casein complex were larger than that of ANS-αs- and ANS-β-casein complexes. Enhancements of fluorescence intensity of complexes of casein components were observed by the addition of KCI or CaCl2. Reason for the enhancement was postulated to be the increase of the quantum yield of the ANS fluorescence caused by the environmental change of ANS binding region of the casein components.Marked increase of sedimentation coefficient of β-casein in the presence of KCl or CaCl2 at 10°C was caused by the addition of ANS. This may be responsible for the stimulation of the Ca-dependent precipitation of β-casein by the addition of ANS.It was found that αs · κ-association was prevented by ANS and that hydrophobic interaction have an important role for αs · κ-association.

Differential sedimentation study on the dissociation and conformational change in hemoglobin

X-ray studies suggest that hemoglobin undergoes a subunit shift when oxygen is bound. The crystallographic measurements indicate that the oxygenated hemoglobin molecule occupies a slightly smaller volume; thus, an increase in sedimentation coefficient would be predicted upon oxygenation. As a complicating factor, the oxygenated hemoglobin begins to dissociate into dimers and even monomers as the protein concentration is lowered, while the deoxygenated molecule retains its tetrameric structure. Using differential sedimentation techniques, these expected changes have been measured. First, the deoxygenated hemoglobin is shown not to be appreciably dissociated in the concentration range between 0.3 to 50 mg/ml., and the sedimentation data are closely approximated by the expression 1 S = 1 S 20, w 0(1 + kc) . Second, the sedimentation coefficient difference between deoxygenated and oxygenated hemoglobin changes by 0.5 s over this concentration range, behaving as predicted from the known dissociation constants. Finally, at the highest concentrations, the oxygenated hemoglobin sediments more rapidly than the deoxygenated form, as would be predicted from the subunit rearrangement determined from X-ray crystallographic studies.

Alkali Denaturation of Covalently Closed Circular Duplex Deoxyribonucleic Acid

Abstract The alkaline pH range over which the double stranded circular DNA of the replicative form of the bacterial virus, ϕX174, is reversibly denatured has been determined in order to specify accurately the conditions for a preparative procedure for this DNA based on selective alkali denaturation. The denaturation remains entirely reversible to pH 12.6 at an ionic strength of about 0.25 over a range in which the sedimentation coefficient, after an initial drop, more than doubles. The sharp transition above pH 12.6 in which the denaturation becomes completely irreversible is accompanied by only a small further increase in sedimentation coefficient. Sedimentation-pH titration curves have been determined in detail for the replicative form of ϕX174 (molecular weight 3.4 x 106 daltons) and for the circular DNA of a penicillinase plasmid of Staphylococcus aureus (molecular weight 16.5 x 106 daltons). The latter curve is displaced to a lower pH and the two phase nature of the curve in the range of increasing sedimentation coefficient is markedly accentuated.