Effect of single nutrient limitation of poly-beta-hydroxybutyrate molecular weight distribution in alcaligens europhus

The effect of magnesium and phosphate limitation on the molecular weight distribution of poly-beta-hydroxybutyrate (PHB) in Alcaligens europhus in cotinuons culture has been stuied. Conditions of nitrogen limitation both with glucose excess (above ca. 20 g/L) and without excess were investigated Under N-limitation and glucose excess, M(w) decreases when the magnesium content is decreased below 50% (19.7 mg/L) of the basal medium content; this also results in a broadenng of molecular weight distribution (M(w)/M(n)) from 2 to 5 and a decrease in M(w) fron 2 x 10(6) to 0.9 x 10(6). Below 20% of the basal content of magnesium (7.9 mg/L) these two trends were reversed. This behaviour was not observed in the absence of glucose excess, phshate had virtually no effect on PHB M(w) or its distribution, whereas wih no (or little) glucose excess M(w) of the PHB decreased with phosphate concentrations below 50% of the basal level (0.705 g/L). Hence, in continuous or fed-batch cultures, in addition to nitrogen limitation to alklow for PHB accumulation, it is necesary to control both the addition of glucose (no excess) and also to maintain magnesium limitation (ca. 25% of basal medium level, 9.9 mg/L) and phosphate above 50% of he basal level (0.705 g/L). Thus, when broadening of molecular weight destribution (increase in M(w)/M(n)) is observed at the end of fed-batch culture it is probably caused by phosphate limitation and/or glucose excess. (c) 1995 John Wiley & Sons, Inc.     

Optimization of high molecular weight pullulan production by Aureobasidium pullulans in batch fermentations

Of five strains of Aureobasidium pullulans studied, NRRL Y-2311-1 yielded the highest titer (26.2 g/L) of pullulan and formed the lowest amount of melanin-like pigment. Sucrose was superior to glucose as the carbon and energy source on the basis of yield and titer of pullulan produced. Pullulan titer was higher (26.2 vs 5.1 g/L), biomass concentration was lower (6.9 vs 12.7 g/L), and DO was lower (0 vs 60% of saturation) when the fermenter was agitated by a marine propeller compared to Rushton impellers. Pullulan produced by strain NRRL Y-2311-1 ranged in weight-average molar mass (M(w)) from 486 KDa and number-average molar mass (M(n)) from 220 Da on day 1 of growth to 390 KDa and 690 Da on day 6; M(w) declined by about 35% from day 1 to day 3, the day of maximum pullulan titer. For the other strains, the ranges of molar mass on the day of maximum pullulan titer were 338-614 KDa (M(w)) and 100-6820 Da (M(n)).     

Determination of molecular weight and molecular structure of rat-liver pyruvate carboxylase.

The molecular weight of pyruvate carboxylase isolated from pigeon and rat liver mitochondria was examined using analytical ultracentrifugation and electron microscopy. The enzyme molecule appeared as a tetramer with the four subunits arranged at the corners of a square. Sedimentation studies in the analytical ultracentrifuge, extrapolated to infinite dilution, showed the tetramer to have a molecular weight Mc=0r of 280 000 and an So20,w of 12.7 S. The tetramer could be dissociated into trimers and dimers of lower specific enzymic activity by storage at 4 degrees C or incubation at -- 20 degrees C at low protein concentrations. The isolated trimers and dimers had a molecular weight Mc=0r of 210 000 and 140 000, respectively, and an So20,w of 10.85 S and 7.55 S, respectively. Incubation with 2 M urea at 20 degrees C yielded enzymically inactive subunits (Mc=0r = 70 000; So20,w = 4.95 S). The molecular weights (for pyruvate carboxylase and its subunits), as calculated from the subunit diameter observed in the electron microscope, were consistent with the values obtained from sedimentation studies.     

A high molecular weight protease in the cytosol of rat liver. II. Properties of the purified enzyme

The properties of a soluble endoprotease from rat liver were studied. The enzyme was purified in a latent form. It sedimented as a single component with a sedimentation coefficient (S(0)20,w) of 19.8 S. Measurement by quasi-elastic light scattering gave a diffusion coefficient (D(0)20,w) of 2.5 X 10(-7) cm2 X s-1 and an effective hydrodynamic radius of 85 A. The enzyme had an unusually high molecular weight, estimated as 743,000 by sedimentation equilibrium and 722,000 by sedimentation velocity and diffusion measurements and as 760,000 by a recently developed low-angle laser light scattering method. Judging from electron microscopic observation and the calculated frictional and axial ratios, the enzyme molecule is disc-shaped. Analysis of the far-ultraviolet circular dichroic spectrum showed that the enzyme contains 50% alpha-helical, 25% beta-sheet, and 15% unordered structures with 10% beta-turns. The isoelectric point of the enzyme is 5.0. These properties indicate that the purified enzyme is a homogeneous molecule. In addition, the enzyme is a simple protein since it contains no measurable amounts of nucleic acid carbohydrate or lipid.     

Characterization of zinc-depleted alanyl-tRNA synthetase from Escherichia coli: role of zinc.

To evaluate the role of zinc in Escherichia coli alanyl-tRNA synthetase, hydrodynamic measurements and circular dichroism spectra were obtained for the zinc-depleted protein and compared with those of the native enzyme. At a protein concentration of 5 mg ml(-1), pH 7.5, the sedimentation coefficient (s(20,w)) was 6.3 S and was virtually independent of temperature between 10 and 37 degrees C, similar to the results reported for the native form. However, the s(20,w) now decreased significantly as the concentration increased, indicative of a possible change in conformation. The s(20,w) value did not appear to change as the pH was increased to 9.5. In standard buffer with 3.3 M added urea, a single peak with a s(20,w) of 3.6 S was obtained and with 6.6 M added urea, a peak with a s(20,w) of 2.7 S was seen. Added Gd-HCl (6 M) gave a single peak with s(20,w) of 2. 0 S. Like the native form, laser light scattering studies indicated some heterogeneity and a radius of 6.4 nm which was virtually independent of concentration and temperature in the range of 10-37 degrees C. At 25 degrees C, a diffusion coefficient (D(20,w)) of 3.3 x 10(-7) cm(2) s(-1) was obtained. The combination of s(0)(20,w) and D(20,w) yielded a molecular mass of approximately 179 kDa, which is slightly less than that reported for the native dimeric form (186 kDa). The intrinsic viscosity at 25 degrees C was extrapolated to 5. 3 ml g(-1), a value significantly higher than that reported for the native form, which increased with temperature. These results indicate some conformational and flexibility changes from the native to the zinc-depleted form, which may explain differences in activity. Furthermore, urea denaturation experiments demonstrate the role of zinc in stabilization of AlaRS structure.     

The molecular weight of bovine lactoferrin.

The molecular weight of bovine lactoferrin in a neutral solvent (e.g. 0.2 M NaC1, pH 7.8 20 degrees C) has been determined using high-speed sedimentation equilibrium at various concentrations and speeds. Under these conditions the protein behaved as a single thermodynamic component with a weight-average molecular weight [MW]C=0 = 93000 plus or minus 2000 and a second virial coefficient B = 4.3-10(-5) mol-ml(-1)-g(-2). This value of the molecular weight is in good agreement with values previously reported by Groves (J. Am. Chem. Soc. 82, 3345-3350, 1960) and Szuchet and Johnson (Eur. Polym. J. 2, 115-128, 1966); it is, however, in disagreement with the more recent value given by Castellino et al. (J. Biol. Chem. 245, 4269-4275, 1970). The possible reasons for this discrepancy are discussed.     

Effect of molecular mass on antitumor activity of heteropolysaccharide from Poria cocos

A water-soluble heteropolysaccharide ac-PCM0 from Poria cocos was successfully fractionated using a preparative size exclusion chromatography (SEC) column, and their weight-average molecular mass (M(w)) was characterized by analytical SEC combined with laser light scattering (SEC-LLS). The results indicate that the fractions having relatively high M(w) exhibited higher inhibition ratio in vivo antitumor activity than those having M(w) below 3.29 x 10(4). However, the relatively low molecular mass was beneficial to the in vitro antitumor activity. Moreover, ac-PCM0 has a significantly higher enhancement ratio of the body weight than 5-fluorouracil, and its 50% lethal dose is above 1250 mg/kg, indicating a nontoxic nature.     

Isolation and physical studies of the intact supercoiled, the open circular and the linear forms of ColE1-plasmid DNA.

For the study of DNA conformations, conformational transitions, and DNA-protein interactions, covalently closed supercoiled ColE1-plasmid DNA has been purified from cultures of Escherichia coli harboring this plasmid and grown in the presence of chloramphenicol according to the method of D.B. Clewell [J. Bact. 110 (1972)667]. The open circular and linear forms of the plasmid were prepared by digestion of the covalently closed, supercoiled form with pancreatic deoxyribonuclease and EcoRI-restriction endonuclease, respectively. The linear form was found to be very homogeneous by electron microscopy and sedimenting boundary analysis. Its physical properties (s0 20,w=16.3 S,D0 20,W=1.98 X 10(-8) cm2 s-1 and [eta]=2605 ml g-1) have been carefully determined in 0.2 M NaCl, 0.002 M NaPO4 pH 7.0,0.002 M EDTA, at 23 degrees C. Combination of s0 20, w (obtained by quasielastic laser light scattering) gave Ms,D=4.39 x 10(6). This value is in reasonable agreement with the molecular weight from total intensity laser light scattering M=4.30 x 10(6). The covalently closed and open circular forms of the ColE1-plasmid are less homogeneous due to slight cross-contamination and the presence of small amounts of dimers in these preparations. The weight fractions of the various components as determined by boundary analysis or electron microscopy are given together with the average quantities obtained in the same solvent for the supercoiled form ((s0 20,w)w=25.4 S, (D0 20,w)z=2.89 x 10(-8) cm2 s-1, [eta]= 788 ML G-1,Ms,D=4.69 x 10(6) and Mw=4.59 x 10(6)) and the open circular form (s0 20, w)w=20.1 S, (D0 20,w)z=2.45 x 10(-8) cm2 s-1, [eta]=1421 ml g-1,Ms,D=4.37 x 10(6) and Mw=4.15 x 10(6)). Midpoint analysis of the sedimenting boundaries allows unambiguous determination of the sedimentation coefficients of these two forms: s0 20,w=24.5 S and s0 20,w=18.8 S, respectively. Also deduced from total intensity light scattering were radii of gyration Rg (103.5, 134.2 and 186 nm) and second virial coefficients A2 (0.7, 4.8 AND 5.4 x 10(-4) mole ml/g2) for the supercoiled, the open circular and linear forms, respectively. The data presented are discussed in relation to the conformational parameters for the three forms in solution.     

Viscosity and molecular weight of hyaluronic acids.

The intrinsic viscosity ([eta]) and the molecular weight (M) by sedimentation equilibrium were determined for hyaluronic acids of low (M=104--7.2X10(4)) and high (M=3.1X10(5)--1.5X10(6)) molecular weights. Double logarithmic plot of [eta] against M gave different lines for the two groups. The relationship between [eta] and M was [eta]=3.0X10(6)XM1,20 for the former and [eta]=5.7X10(-4)XM0.46 for the latter group. The molecular weight at the point of intersection of the two lines was about 1.5X10(5). The rheological behavior of the hyaluronic acids below M=2.1X10(4), for which the value of reduced viscosity was independent of concentration, was different from that of the hyaluronic acids above M=5.1X10(4), for which the value of reduced viscosity increased with concentration.     

Effects of molecular weight on the miscibility and properties of polyurethane/benzyl starch semi-interpenetrating polymer networks

We successfully prepared a series of semi-interpenetrating polymer networks (semi-IPNs) from castor oil-based polyurethane (PU) and 20 wt % benzyl starch (BS) with different weight-average molecular weights (M(w)), coded as the PU/BS films. The M(w) values of a series of BSs were determined by size-exclusion chromatography combined with laser light scattering. The effects of the BS M(w) on the miscibility and properties of the resulting PU/BS films were investigated using reflection Fourier transform infrared spectroscopy, differential scanning calorimetry, dynamic mechanical thermal analysis, scanning electron microscopy, optical microscopy, ultraviolet-visible spectroscopy, and tensile testing. The results revealed that the PU/BS films possessed much higher optical transmittance and tensile strength than the pure PU film. Interestingly, with a decrease of the BS M(w) from 1.69 x 10(7) to 5.70 x 10(5), the optical transmittance, tensile strength, and elongation at break of the PU/BS films increased from 82% to 89%, from 11.7 to 15.7 MPa, and from 121% to 180%, respectively. Therefore, the M(w) of BS plays an important role in the improvement of the miscibility and properties of the semi-IPN materials. On the basis of the analysis of the miscibility and the morphology of the PU/BS films, the interaction between the PU and the BS with relatively low M(w) was stronger than that with high M(w).     

Structure and synthesis of a lipid-containing bacteriophage. The molecular weight and other physical properties of bacterophage PM2.

Several physical and chemical parameters of bacteriophage PM2 have been measured. The sedimentation constant was determined to be s-20,w=293 S. The buoyant density in sucrose at 20 degrees C was 1.24 g cm+-3 and in CsCl at 25 degrees C was 1.29 g cm-3. The high-speed equilibrium centrifugation method of Yphantis (1964) was used to measure the molecular weight of PM2. The necessary auxiliary parameters were also determined. A value of 0.771 plus or minus 0.005 cm-3 g-1 for the apparent specific volume at constant chemical potential in 1 M sodiium chloride has been obtained by pycnometry; the viral concentration was determined using the absorption coefficient at 260 nm (4.60 plus or minus 0.10 cm-2 mg-1), which in turn was calculated from the phosphorous content of the virus (17.89 plus or minus 0.28 mu-g of P per mg dry weight dry weight of virus). The molecular weight of PM2 determined with these parameters is (44.1 plus or minus 1.2 x 10-6). From the phosphorous content of the virus, the percentage of phosphorous known to be in its DNA (Camerini-Otero and Franklin, 1972), and the molecular weight of the bacteriophage, we have calculated a molecular weight for PM2 DNA of 6.26 x 10-6, which confirms values determined using empirical relationships.     

Pre-formed xyloglucans and xylans increase in molecular weight in three distinct compartments of a maize cell-suspension culture

Cultured cells of maize ( Zea mays L.) were pulse-labelled with l-[1-(3)H]arabinose (Ara) and then monitored for 7 days. The (3)H-hemicelluloses present in three compartments (protoplasm, cell wall and culture medium) were size-fractionated and the fractions assayed for [(3)H]xyloglucans and [(3)H]xylans. Protoplasmic [(3)H]xylans and [(3)H]xyloglucans initially (15 min after [(3)H]Ara-feeding) had weight-average relative molecular masses ( M(w)) approximately 0.5x10(6) and 0.3x10(6), respectively, both rising to 2x10(6) by 30 min. Thus, newly formed hemicellulose molecules were joined to other polymers, or to each other, presumably within Golgi vesicles. New (3)H-hemicelluloses very rapidly bound to the cell wall; however, after 1 day, some [(3)H]xyloglucan and [(3)H]xylan was sloughed from the wall into the medium. The wall-bound [(3)H]xyloglucans were present in the form of extremely large complexes, of M(w)>17x10(6), even as early as 15 min after [(3)H]Ara-feeding. This M(w) is >70-fold greater than that observed by similar methods in cultures of a dicotyledon ( Rosa sp.). Thus, during wall-binding, newly secreted xyloglucans greatly increased in size, possibly by transglucosylation. Some modest degradation (trimming) of wall-bound [(3)H]xyloglucan occurred later. The earliest wall-bound [(3)H]xylan had M(w) approximately 2x10(6), similar to the protoplasmic [(3)H]xylan; this increased to approximately 4x10(6) by 6 h. For the first 2 days after [(3)H]Ara-feeding, the soluble extracellular (3)H-hemicelluloses present in the culture medium had M(w) approximately 1x10(6)-2x10(6), comparable to the protoplasmic hemicelluloses. However, between 2 and 3 days after [(3)H]Ara-feeding, the M(w) of the soluble extracellular [(3)H]xylans increased abruptly to approximately 10x10(6); the soluble extracellular [(3)H]xyloglucans underwent a similar but more gradual increase in M(w). Maize (3)H-hemicelluloses thus underwent increases in M(w) in three episodes: (i) intra-protoplasmically, (ii) during wall-binding (especially xyloglucans), and (iii) after sloughing into the medium. Possible mechanisms and roles of these increases are discussed.     

Pigeon liver amidophosphoribosyltransferase. Ligand-induced alterations in molecular and kinetic properties.

Amidophosphoribosyltransferase (EC 2.4.2.14) has been partially purified from pigeon liver, and ligand-induced alterations in molecular and kinetic properties have been studied. In Tris-HCl buffer the predominant form of the enzyme has an s20,w of 5.9 +/- 0.7, Stokes radium of 42 A, and estimated molecular weight of 102,000. Incubation with phosphoribosylpyrophosphate (PP-ribose-P) results in an increase in the s20,w to 7.9 +/- 0.6, Stokes radius to 53 A, and estimated molecular weight to 172,000. Incubation of this larger form with purine ribonucleotides leads to a decrease in the molecular weight of amidophosphoribosyltransferase that is proportional to the concentration of purine ribonucleotide. Purine ribonucleotides produce sigmoidal kinetics with respect to the substrate PP-ribose-P, with Hill coefficients of 1.4 to 1.6 and 1.8 to 2.0 in the presence of AMP and GMP, respectively. Incubation with 0.6 M KCl leads to sigmoidal kinetics. Hill coefficient of 1.8 and dissociation of the larger form of amidophosphoribosyltransferase. Inorganic phosphate has complex effects upon the enzyme. In 25 mM potassium phosphate buffer the enzyme aggregates to a large form with an s20,w of 8.3 +/- 0.2, Stokes radius of 53 A, and estimated molecular weight of 181,000. Inorganic phosphate and PP-ribose-P both stabilize the enzyme to storage in vitro at 4 degrees. However, inorganic phosphate is 4 times more effective than PP-ribose-P in preventing inactivation of the enzyme by sodium dodecyl sulfate. Inorganic phosphate produces sigmoidal kinetics with respect to PP-ribose-P, Hill coefficient of 1.5. The interaction coefficients for AMP and GMP are reduced from 1.8 to 1.2 and 2.2 to 1.4, respectively, in the presence of 25 mM potassium phosphate. It is concluded that pigeon liver amidophosphoribosyltransferase is a complex allosteric protein whose activity is regulated by a series of conformational changes induced by a number of ligands.     

Molecular weight of Eudistylia vancouveri chlorocruorin and its subunits.

The chlorocruorin of the marine polychaete Eudistylia vancouveri has a molecular weight of 3.1-10(6) and a sedimentation coefficient (S020, w) of about 57 S at pH 8.0 in the presence of 0.01 M Mg2+. The quaternary structure of this pigment is unaffected by pH between 6.0 and 11.5 in the presence of 0.01 M Mg2+ whereas in 0l01 M EDTA, the pigment begins to dissociate above pH 9.0 into smaller submultiples. The chlorocruorin can be converted into subunits with molecular weights of about 14 000-15 000 and 30 000 as determined by sodium dodecyl sulfate-gel electrophoresis and 14 000-15 000 as measured by gel chromatography of the carboxy-methylated derivative in 8 M urea, 0.1 M 2-mercaptoethanol, or by sedimentation equilibrium in 6 M guanidine-HCl and 0.1 M 2-mercaptoethanol. The pigment contains 0.212 +/- 0.008% iron corresponding to 1 g atom iron per 26 300 g chlorocruorin. The amino acid composition of this pigment is reported. The subunit structure of Eudistylia chlorocruorin and the polymeric annelid hemoglobins are similar in many respects.     

The purification and properties of isocitrate lyase from Chlorella

1. Isocitrate lyase (threo-d(s)-isocitrate glyoxylate-lyase, EC 4.1.3.1) has been purified from acetate-adapted cells of Chlorella pyrenoidosa. 2. The final preparation was homogeneous by the criteria of sedimentation, diffusion and polyacrylamide-gel electrophoresis. 3. The sedimentation coefficient (S(20,w)) was 9.04x10(-13)sec. and the diffusion coefficient (D(20,w)) 4.62x10(-7)cm.(2)/sec.; from these values the molecular weight of the enzyme was calculated to be 170000 and its Stokes radius to be 4.63x10(-7)cm. 4. The elution of the enzyme from Sephadex G-100 was studied and estimates of molecular weight and Stokes radius were obtained from the elution data. 5. The turnover number of the enzyme was 5950moles of glyoxylate formed/min./mole of enzyme at 30 degrees . 6. With threo-d(s)(+)-isocitrate as substrate, the K(m) of the enzyme was 0.023mm.     

Studies on Transformations of Hemophilus influenzae : II. The molecular weight of transforming DNA by sedimentation and diffusion measurements

The sedimentation and diffusion coefficients have been determined for Hemophilus influenzae transforming activity and DNA using P³²-labeled DNA. The methods employed the Spinco fixed boundary separation cell for measurements of the sedimentation coefficient and the Northrop-Anson diffusion cell to determine the diffusion coefficient. There was a very close correlation between the amount of DNA and transforming activity sedimented or diffused. The sedimentation coefficient (s_20°), for both biological activity and DNA was 27 and the diffusion coefficient (D_20°) 1 x 10^-8 cm²/sec. The molecular weight calculated from these coefficients gave a value of 16 million. There was no difference in the sedimentation coefficients for the two unlinked markers, streptomycin and erythromycin resistance, and the diffusion coefficients for single markers or the linked markers, streptomycin and cathomycin, were the same.     

A method for the measurement of the sedimentation coefficient and molecular weight of microgram quantities of proteins in 6 M guanidine hydrochloride

A technique has been perfected for measuring the sedimentation coefficient of microgram quantities of a reduced protein in 6 M guanidine hydrochloride. The protein is sedimented through a gradient of 5-8 M guanidine-HCl in the presence of dithiothreitol in a SW 50.1 swinging-bucket rotor. Run conditions are calibrated by a simultaneous measurement using a single reference protein. Thus, the need for running a calibration curve involving several standard proteins simultaneously with a sample is eliminated. Because of the trace quantity of protein used, the technique yields an estimate of the sedimentation coefficient at zero concentration (s0) directly without extrapolation. Since s0 is a function of the molecular weight of a reduced protein in this solvent, the method also allows an estimate of the subunit molecular weight of the protein. The results of the application of the method to known proteins are reported.     

Mustard seed meal mixtures: management of Meloidogyne incognita on pepper and potential phytotoxicity

Meals produced when oil is extracted from seeds in the Brassicaceae have been shown to suppress weeds and soilborne pathogens. These seed meals are commonly used individually as soil amendments; the goal of this research was to evaluate seed meal mixes of Brassica juncea (Bj) and Sinapis alba (Sa) against Meloidogyne incognita. Seed meals from Bj 'Pacific Gold' and Sa 'IdaGold' were tested alone and in combinations to determine rates and application times that would suppress M. incognita on pepper (Capsicum annuum) without phytotoxicity. Rates of soil application (% w/w) for the phytotoxicity study were: 0.5 Sa, 0.2 Bj, 0.25 Sa + 0.25 Bj, 0.375 Sa + 0.125 Bj, 0.125 Sa + 0.375 Bj, and 0, applied 0 - 5 weeks before transplant. Overall, 0.2% Bj was the least toxic meal to pepper seedlings. By comparison, 0.5% S. alba seed meal did not reduce lettuce (Lactuca sativa) seed germination at week 0, but all seed meal treatments containing B. juncea prevented or significantly reduced germination at week 0. The seed meals did not affect lettuce seed germination at weeks 1-5, but hypocotyl growth was reduced by all except 0.2% Bj at weeks 1, 4 and 5. Brassica juncea and Sa meals were tested for M. incognita suppression at 0.2, 0.15, 0.1 and 0.05%; mixtures were 0.1% Sa + 0.1% Bj, 0.15% Sa + 0.05% Bj, and 0.05% Sa + 0.15% Bj. All treatments were applied 2 weeks before transplant. The 0.2% Bj and 0.05% Sa + 0.15% Bj treatments overall had the longest shoots and highest fresh weights. Eggs per g root were lowest with 0.1 - 0.2% Bj amendments and the seed meal mixtures. The results indicate that Bj and some Bj + Sa mixtures can be applied close to transplant to suppress M. incognita populations on pepper; consequently, a seed meal mixture could be selected to provide activity against more than one pest or pathogen. For pepper, care should be taken in formulating mixtures so that Sa rates are low compared to Bj.     

A light-scattering study of the effect of calcium chloride on the molecular weight of Busycon hemocyanin.

Solutions of Busycon canaliculatum have been studied by light scattering. In 0.05 M Trizma buffer +0.1 M NaCl at pH 7.0 at 14 degrees, the weight-average molecular weight is 8.9 X 10(6). In the presence of added CaCl2 (0.02 M), the molecular weight of the protein increases to 10.7 X 10(6), and the second virial coefficient is reduced. At pH 9.95, the molecular weights with and without 0.02 M CaCl2, are 3.7 X 10(6) and 1.3 X 10(6), respectively; and the effect of Ca++ in reducing the second virial coefficient is much greater than at pH 7.0. These results can be understood on the basis that at pH 7.0, ca++ increases the association of hemocyanin, by binding and intermolecular linkage through the carboxyl groups of protein side chains. At pH 9.95, amino groups are deprotonated and therefore also become available for Ca++ binding. The relative effect of Ca++ in enhancing the association of hemocyanin therefore becomes greater at the higher pH.     

Molecular weight standards for calibration of gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis: ferritin and apoferritin

Ferritin and apoferritin are widely used for the calibration of gel filtration columns and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and are commercially offered for these purposes as part of molecular weight calibration kits. Many of the reported applications are severely in error as presented in leading references and application manuals. The manufacturers have based their recommendations on incorrect physicochemical parameters in the literature and incorrect or inadmissible assumptions about the protein subunit composition and architecture and have not taken into account the unusual resistance of these proteins to denaturation in SDS. Here the relevant physicochemical parameters of horse spleen apoferritin as reported in the literature are critically reevaluated and the best current estimates are identified as the following: weight average molecular weight of apoferritin, Mw = 481,200; molecular weight of subunits, major subunit, ML = 19,889; minor subunit, MH = 22,200; apparent specific volumes in 0.02 M acetate buffer, pH 5.5, and 0.1 M NaCl, phi = 0.721 ml g-1 and phi' = 0.743 ml g-1; partial specific volume at 20 degrees C, v = 0.738 ml g-1; viscosimetric molar volume, M[n] = 1.78 X 10(6) ml mol-1; Stokes radius, RSt = 67.1 A; viscosimetric radius, Rvis = 65.6 A; sedimentation coefficient S degrees 20, w = 16.6 S; translational diffusion coefficient, D20, w = 3.24 X 10(-7) cm2 s-1. Recommendations are provided for proper application of ferritin and apoferritin for calibration purposes in gel filtration and SDS-polyacrylamide gel electrophoresis.