Specific Refractive Increment Research Articles

SEDNTERP: a calculation and database utility to aid interpretation of analytical ultracentrifugation and light scattering data.

Proper interpretation of analytical ultracentrifugation (AUC) data for purified proteins requires ancillary information and calculations to account for factors such as buoyancy, buffer viscosity, hydration, and temperature. The utility program SEDNTERP has been widely used by the AUC community for this purpose since its introduction in the mid-1990s. Recent extensions to this program (1) allow it to incorporate data from diffusion as well as AUC experiments; and (2) allow it to calculate the refractive index of buffer solutions (based on the solute composition of the buffer), as well as the specific refractive increment (dn/dc) of proteins based on their composition. These two extensions should be quite useful to the light scattering community as well as helpful for AUC users. The latest version also adds new terms to the partial specific volume calculations which should improve the accuracy, particularly for smaller proteins and peptides, and can calculate the viscosity of buffers containing heavy isotopes of water. It also uses newer, more accurate equations for the density of water and for the hydrodynamic properties of rods and disks. This article will summarize and review all the equations used in the current program version and the scientific background behind them. It will tabulate the values used to calculate the partial specific volume and dn/dc, as well as the polynomial coefficients used in calculating the buffer density and viscosity (most of which have not been previously published), as well as the new ones used in calculating the buffer refractive index.

Protein standardization II: Dry mass determination procedure for the determination of the dry mass of a pure protein preparation.

A procedure has been developed for the determination of the dry mass of a pure protein preparation dissolved in one electrolyte. The procedure not only renders the concentration (in g/l), but additionally gives the partial specific volume of the protein (in ml/g). The latter is an important parameter for the characterization of a specific protein. Furthermore, the importance of extensive dialysis against one electrolyte is discussed. For the drying of human serum proteins it is clearly shown that KCl is preferred to NaCl due to its stable temperature curve. By observing the parallel fluctuations in the weight of the empty vials during drying, an average correction factor is introduced, which greatly minimizes these changes. The assay principle is discussed from a mathematical as well as from a practical point of view. A detailed procedure is described and the final results for three primary pure proteins (prealbumin, orosomucoid and transferrin) are presented. Finally, important parameters such as the wavelength of maximum absorbance, the absorption coefficient and the specific refractive increment are discussed and values for the three proteins are presented. When these parameters have been established the future determination of concentration and the characterization of pure protein solutions are greatly facilitated. These procedures were important tools for ascribing mass values for prealbumin, orosomucoid and transferrin to the international reference preparation CRM 470.

Effect of fluctuations on the refractive indices of some pure liquids

This work describes an indirect experimental method for the determination of the specific refractive increments (∂n/∂T)ϱ. Measurements carried out on water and several organic liquids are reported. Different relations between density and refractive index of pure liquids are used. The results prove the superiority of the relation suggested by us.

Analytic ultracentrifuge calibration and determination of lipoprotein-specific refractive increments.

Accurate quantification of the major classes and subfractions of human serum lipoproteins is an important analytical need in the characterization and evaluation of therapy of lipid and lipoprotein abnormalities. For calibrating the analytic ultracentrifuge (AnUC), we routinely use a Beckman calibration wedge cell with parallel scribed lines 1 cm apart. Such a cell gives a rectangular pattern in the schlieren diagram, which determines magnification and also provides an area corresponding to an invariant refractive increment. We have independently validated this wedge calibration cell using a special boundary-forming cell in which 1.174% sucrose is overlayered with distilled water. Comparing wedge cell area with extrapolated zero time boundary area refractive increment gives agreement to within less than 1%, corresponding to a refractive increment error of +/- 0.00002 delta n. Complete calibration for AnUC analysis of lipoproteins also requires accurate determination of the specific refractive increments (SRI) of the major lipoprotein classes, namely low density lipoprotein (LDL) and high density lipoprotein (HDL). These are measured in the density in which they are analyzed, i.e., 1.061 g/ml for LDL and 1.200 g/ml for HDL. Five fresh serum samples were fractionated for total LDL and total HDL and their SRI determined. Total lipoprotein mass was determined using precise CHN elemental analysis and compositional analyses. The results yielded corrected SRI of 0.00142 and 0.00135 delta n/g/100 ml for LDL and HDL. Thus, our current values using 0.00154 and 0.00149 delta n/g/100 ml underestimate LDL and HDL by 9% and 11%. Corrections of all previous LDL and HDL AnUC data can be made using appropriate factors of 1.087 and 1.106.

Light-scattering and specific refractive increment behavior of amylose and dextran in methyl sulfoxide-water

Light-scattering measurements on polysaccharides in methyl sulfoxide or methyl sulfoxide-water are most reliable when made on a dialyzed solution-solvent system and when proper technique is used to remove the “micro gel” that seems unavoidably present when dried polysaccharide is dissolved in solvent. Discrepancies in published molecular weight-intrinsic viscosity relationships for amylose in methyl sulfoxide appear to be caused in part by differences the preparation and treatment of samples and possibly by differences in botanical source. The specific refractive increment of dextran is not a linear function of solvent composition in methyl sulfoxide-water. Measurements at osmotic equilibrium indicate that, over a moderate range of composition in methyl sulfoxide-water, dextran preferentially associates with about 2.5 molecules of water per glucopyranosyl residue of the polymer. Effects of long-term storage, sample treatment, and botanical source upon the specific rotation of amylose are examined.

Physical and chemical properties of human plasma alpha2-macroglobulin.

Alpha2-M (alpha2-macroglobulin) was purified from human plasma by two different procedures. As well as having no detectable impurities by the usual criteria for testing the homogeneity of protein preparations, these alpha2M preparations showed a single component, after reduction in urea, of 185000 daltons by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The molecular weight of the alpha2M was found to be 718000 by sedimentation equilibrium experiments using the gravimetrically determined -v of 0.731 ml/g. The interaction of several proteinases with alpha2M was studied by using a novel discontinuous polyacrylamide-gel system, which showed clear separation of the enzyme-complexed alpha2M from the free alpha2M. These studies indicated that urokinase, as well as trypsin, chymotrypsin, plasmin and thrombin forms complexes with alphaM. The cleavage of the 185000-dalton subunit to a 85000-dalton species on interaction of trypsin with alpha2M was demonstrated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis after reduction of the alpha2M-trypsin complex in urea. The amino acid composition, carbohydrate content, absorption coefficient at 280 nm, the specific refractive increment and the sedimentation coefficient for these alpha2M preparations were measured. The stability of the trypsin-binding activity of the alpha2M preparations was also studied under several storage situations.

New differential Bottcher-Onsager method used to determine polarizability and apparent radius of tungstosilicate (SiO4(WO3)124-)

A differential form for the Bottcher--Onsager law is derived. Based on this form, a new method is presented for determining the polarizability and apparent radius of the molecules of a solute from measurements of the temperature variation of the density and specific refractive increment of its solutions. The method is particularly applicable to solutes which are only slightly soluble in a given solvent. The density and specific refractive increment of aqueous solutions of tungstosilicic acid have been measured at four temperatures: nominally, 20, 25, 35, and 45/sup 0/C. The densities are found to be linear functions of concentration in the range 0 to 0.03 M. From the density measurements, the apparent molar volume of H/sub 4/SiO/sub 4/(WO/sub 3/)/sub 12/ at each temperature is determined. The specific refractive increment is determined for radiation at 589 m..mu... Using our differential method, we find ..cap alpha../sub 2/ = 50 +- 5 A/sup 3/ and a/sub 2/ = 3.2 +- 0.2 A, where ..cap alpha../sub 2/ and a/sub 2/ are the polarizability and effective radius, respectively, of SiO/sub 4/(WO/sub 3/)/sub 12//sup 4 -/.

Temperature dependence of optical properties of aqueous dispersions of phosphatidylcholine

Aqueous dispersions of dipalmitoyl phosphatidyl choline exhibit a sharp decrease in turbidity at the crystal-liquid phase transition temperature of 41°C. The intensity of light scattered at 45°, 90°, and 135° also undergoes a sharp drop at the same temperature. Similarly, the refractive index of such dispersions decreases abruptly with the phase transition. Employing the relationship between light scattering intensity and specific refractive increment, it can be shown that about one half of the change in absorbancy and scattering are accounted for by the change in refractive index. The change in refractive index can be entirely accounted for by the known expansion and corresponding decrease in density of the bilayer. That part of the observed change in scattering and turbidity which is not accounted for by the observed change in average refractive index is apparently due to a decrease in the anisotropy of the bilayer during the melting process. Calculations based on a model which, although oversimplified, is consistent with the known thinning of the bilayer during the melting process, give quantitative agreement with experimental results. Below the phase transition temperature other changes in optical properties are observed; near 32°C, the light scattering envelope changes and the turbidity of dispersions drops markedly. The average refractive index remains constant in this region. For this and other reasons, it is postulated that these pre-transition changes indicate an aggregation-disaggregation phenomenon.

Sedimentation equilibrium in nonideal heterogeneous systems. I. Fundamental equations for heterogeneous solute systems and some preliminary results

AbstractThe sedimentation‐equilibrium method is extended to treat nonideal solutions of heterogeneous macromolecules. The solute is assumed to be heterogeneous not only in molecular weight but also in other quantities such as partial specific volume, second virial coefficient and specific refractive increment. General expressions for various observable molecular weights, especially for weight‐average, z‐average, and number‐average molecular weights, are derived. Their dependences on sedimentation parameter and solute concentration are discussed in detail. For the extrapolation of observable molecular weights, giving a type of weight‐average, and z‐average, to infinite dilution to estimate the molecular weight and the second virial coefficient, average concentration is superior as a concentration variable to original concentration. The plots of observable molecular weight versus average concentration are usually less influenced by the choice of the sedimentation parameter, especially of rotor speed. The general expressions are applied to a few special cases; monodisperse polymer, polydisperse homologous polymer, and polymer blend. The results are compared with experiments on a monodisperse, polystyrene, a polydisperse poly(methyl methacrylate), and a mixture of the two polymers, all in 2‐butanone at 25°C. The agreement between the theory and experiments is satisfactory.

Sedimentation equilibrium and other physicochemical studies on the lysine-rich fraction of calf thymus histones.

1. The lysine-rich fraction (Ia+Ib, or f1) of calf thymus histones was isolated as the sulphate by acid extraction. 2. Sedimentation-equilibrium measurements with interference optics showed that this fraction was monodisperse with a molecular weight of 19500+/-2000. 3. The ;apparent molecular weight' calculated from the sedimentation-equilibrium studies varied markedly with concentration. The large second virial coefficient implied by such variation was attributed to the very high charge/mass ratio of this relatively small protein. Estimates of the charge were made from the values of this virial coefficient. 4. The very large value of the virial coefficient explains anomalies in the earlier reports of the molecular weight of this histone and also why the z-average molecular weight can appear to be lower than the weight-average molecular weight. 5. The differences of the specific refractive increments, and the partial specific volumes, between dialysed and undialysed solutions of this histone fraction could also be attributed to its high molecular charge, which was estimated from these differences and agreed, within the expected limits, with the value deduced from the second virial coefficient. 6. Sedimentation-velocity measurements combined with the known molecular weight imply that lysine-rich histone has a high frictional ratio and an extended shape. Optical-rotatory-dispersion measurements indicated that it had a low helical content.

Some physical properties of purified fraction I protein from spinach chloroplasts

Fraction I protein, from spinach chloroplasts and homogeneous by electrophoretic and sedimentation velocity criteria, had an uncorrected specific refractive increment of 1.69 × 10 −4 ml/mg protein at 10.0 ° and 546 mμ. Its specific extinction coefficient was 1.82 at 1 mg/ml in pH 8.0, 0.001 m Tris-chloride for a 1-cm light path. Moving boundary electrophoresis of this protein at about 4 ° yielded mobilities from −3.2 to −6.2 × 10 −5 cm 2 volt −1 sec −1 at pH 6.3–9.5, which, on extrapolation, indicated an isoelectric point around pH 4.3–5.5. The sedimentation coefficient decreased with increasing protein concentration above 0.3 mg/ml and was 18.7S when extrapolated to zero concentration. At less than 0.3 mg/ml, the sedimentation coefficient decreased with decreasing concentration, suggesting the dissociation of the protein. The apparent diffusion coefficient was 3.01 × 10 −7 cm 2 sec −1, which when combined with the extrapolated sedimentation coefficient gave a molecular weight of 559,000. Sedimentation equilibrium experiments gave a limiting molecular weight at the meniscus of 475,000 ± 11,000 and suggested the presence of a 545,000 molecular weight component. An attempt was made to explain the 17% discrepancy between the molecular weights from sedimentation velocity runs and that from sedimentation equilibrium runs

Refraction and absorption of light in bacterial suspensions

The apparent average refractive indices of three types of microorganism in liquid suspension have been estimated by two methods. In the first, the refractive index of the aqueous suspension is measured in an interferometer, the bacterial cell volume fraction is estimated from the dilution of an added inert solute, and the refractive index of the disperse phase is calculated with the aid of a mixture rule. In the second method, optical density spectra of suspensions in several concentrated solutions of nonpenetrating solutes are measured as a function of the refractive index,n1, of the solution. The value ofn1 when the optical density,E, has its smallest value is equated to the average cell refractive index and the constants of the parabolaE (n1) are related to the heterogeneity of the cell population in respect to refractive index. The preliminary results obtained by the two methods are in satisfactory agreement and are consistent with the specific refractive increments of protein and nucleic acid and their concentrations in the cell. It is pointed out that measurements of optical density spectra can also give useful information concerning the number and size of cells in a suspension and their optical dispersion constants. To obtain such information, however, a spectrophotometer specially designed to exclude forward-scattered light must be used and an acceptable theory must be available. Finally, the apparent spectral absorption of “clarified” cell suspensions is presented and compared with spectra obtained by other methods. It is pointed out that because of refractive heterodispersity even “clarified” suspensions are turbid, so that this method does not provide a completely satisfactory technique for cell spectrophotometry.

On the colligative nature of the specific refractive increment for statistical copolymers

AbstractThe specific refractive increment for statistical copolymers is shown to be colligative in nature for copolymers of styrene–methyl methacrylate thus giving direct evidence to support the assumption of Stockmayer et al. on the theory for light scattering in copolymers. The determination of the mole fraction of mer composition in copolymers by specific refractive increment measurements is shown to be a useful technique and nearly as precise as chemical analysis for carbon. The technique is restricted to soluble systems and is limited by the resolution of the measuring instrument.

Determination of the dry mass of rat liver cells to study the protective mechanism of cysteamine against whole-body X-irradiation

An interference microscope was used in Cytointerferometric measurements of the dry mass of cytoplasm, heterochromatin, and nucleolus of fixed rat hepatic cells in tissue sections. The specific refractive increment of protein in glycerine was employed in dry mass calculations on cells from normal control, irradiated control, and cysteamine-protected rats. The values for heterochromatin and nucleolus in the liver cells of cysteamine-protected rats were considerably higher than in cells of irradiated controls. (C.H.)

The concentration and purification of poliomyelitis virus by the use of nucleic acid precipitation

The precipitation of poliovirus by nucleic acid at low pH represents a new, highly satisfactory method for the concentration of virus from large volumes of tissue culture filtrate. This method has been used as a first step in a procedure for the purification of poliovirus. Virtually pure preparations so obtained have permitted the redetermination of the extinction coefficient of poliovirus ( E 260 190 = 81.6) and the determination of the specific refractive increment of the virus ( Δ n = 0.00174). Sufficient quantities of the virus became available through the use of this procedure for the determination of the elementary composition of the virus. Purified preparations of poliovirus obtained by the use of this method were inactivated with formaldehyde and were found to induce neutralizing antibodies in animals and man.

Studies in histochemistry. LVII. Determination of the total dry mass of human erythrocytes by interference microscopy and x-ray microradiography.

The total dry mass of human erythrocytes was determined by both interference microscopy and x-ray microradiography. The determination of mass per unit area, and calculation of total dry mass per cell were simplified by changing the shape of the cells to spheres which were then flattened to discs of constant thickness when smeared on glass slides for measurement of fixed cells by interferometry, and to oblate spheroids when smeared on parlodion-coated slides for measurement of fixed cells by x-ray absorption. From x-ray measurements of 100 smeared and alcohol-fixed cells a mean dry mass per cell of 33.7 x 10(-12) g was obtained. Interference measurements of 100 fresh cells suspended in isotonic saline gave a mean value of 32.4 x 10(-12)g while interference measurement of 100 smeared and alcohol-fixed cells gave a mean value of 30.8 x 10(-12)g. The first two values compare well with a mean corpuscular hemoglobin of 31.2 x 10(-12)g, obtained from determinations of erythrocyte count and hemoglobin, since 95 per cent of the dry mass of the cell is hemoglobin. The difference in interference values between the fixed and fresh cells is possibly due to a difference between the specific refractive increment of alcohol-denatured hemoglobin and that of the unmodified substance. The value for the latter was used since that of the former is unknown.

Interaction between albumin, lecithin and cephalin.

LECITHINS and cephalins form more or less stable combinations with proteins1,2. These are believed to be the result of lipid–protein interaction3. Provided the method is applicable, information regarding the interaction may be obtained by an electrophoretic method4 used for the study of interaction between dextrans and proteins. Essentially, this method uses the Antweiler micro-Tiselius apparatus to obtain spikes, at various pH's and concentrations, of the two interacting substances and of their mixtures. Plotting the areas under the spikes against concentration enables one to determine a function of α, the specific refractive increment, and sometimes to decide as to whether the mixture of the two components has resulted in interaction between them.

Thermal denaturation of lobster hemocyanin

1. 1. A procedure for the determination of heat denaturation of hemocyanin by means of changes in specific refractive increment has been presented. The method possesses the advantage of requiring a small volume (0.1 ml) of solution for analysis. 2. 2. Denaturation was found to follow first order kinetics and yielded an activation energy of 80,960 cal mol −1. 3. 3. The rate of denaturation of hemocyanin at constant temperature varied inversely as the first power of the hydrogen ion concentration, suggesting a dissociation of one proton in the range of pH studied. 4. 4. Thermodynamic analogues based upon the theory of absolute reaction rates were calculated from the data. Approximate values of ΔF ∗, ΔH ∗, and ΔS ∗ were found to be approximately 20,700 cal mol −1, 80,000 cal mol −1 and 175 cal degree −1, respectively.

An Integration Method for the Interference Microscope

ABSTRACT A method is described of measuring the integrated phase retardation of an object with a Baker interference microscope. With certain limitations, the total dry mass of the object can be derived from this figure. This method of ‘weighing’ objects is of particular value in measuring the growth of single cells, since it is very sensitive and can be used on a living cell without damaging it. The method consists of restricting the field round the object with an aperture of known area and then measuring the total retardation within this field due to the presence of the object. The compensator is adjusted until there is an equal light intensity in the field with and without the presence of a quarter-wave plate. A similar adjustment is made with the object moved out of the field. The difference between the two compensator settings gives the integrated area retardation due to the object. The quarter-wave plate is motor-driven, and the light intensity is measured by a photomultiplier and presented on a cathode ray tube. The instrument is sensitive to a change of about 5 × 10_ 14 g in the dry weight of a biological object. For an object of known specific refractive increment and mounted in water, the total dry mass can be derived from the retardation without the dimensions of the object being known. There is, however, a severe limitation with this method of integration— that the maximum retardation of the object must not exceed about 45 º or λ/8. This can be shown by theory, and has also been confirmed by model experiments with small celloidin spheres. Most biological objects have larger retardations in water, so it is necessary to mount them in media of higher refractive index. In this case, the volume of the object must also be known for the dry mass calculation. Although this limits the use of the method, it is important in measuring the growth of a cell to know the volume as well as the dry mass. An example is shown of the application of this method in measuring the growth of a fission yeast, Schizosaccharomyces pombe. Curves are given showing the changes in dry mass, volume, and concentration of a single growing and dividing cell of this yeast.

A comparison of the properties of various preparations of sodium alginate

AbstractFifteen specimens of sodium alginate from various brown algae, including species of Laminaria, Fucus and Ascophyllum, have been prepared in the laboratory or obtained from commercial sources. Tests on these samples included measurements of optical rotation, ionic mobility, rate of sedimentation, viscosity and content of sulphated ash. Most specimens were homogeneous by electrophoresis and in the ultracentrifuge. The content of alginate of the samples varied from 61.7 to 99.6%. Rate of sedimentation was strongly dependent on concentration. Electrophoretic mobilities varied only slightly. Viscosity varied much more widely than other physical properties and was the best criterion of state of polymerization. Fucoidin was detected as impurity in some preparations.From the best preparations examined the following values were obtained: [α]d20° = −133°to −140°; μ = 2.9 to 3.1 × 10−4 cm.2 sec.−1 volt−1 at 25°, pH 5.5, I 0.05; s40 = 1.3 to 1.6 S (c = 0.5%); sulphated ash = 34.0 to 35.9%. Viscosity varied from 2 to 20 centistokes at 25°in 0°1n‐NaCl (c = 0.25% w/w). Values for [η] varied from 3.6 to 19.2 corresponding to a degree of polymerization of 210 to 1,100 and particle weight of 42,000 to 222,000. The specific refractive increment of sodium alginate was found to be 0.160 g.−1 cm.3.