Prediction of retention volumes and UV spectra of peptides in reversed phase HPLC

A method for calculating retention volumes of linear peptides with known primary structures and the values of their UV absorption at chosen wavelengths in reversed phase HPLC are described. These parameters are calculated for every peptide on the basis of the contributions of its amino acid residues determining its degree of retention and its UV spectrum. The contribution values are experimentally found from chromatograms of the free amino acids obtained by multiwavelength photometric detection under the conditions of the peptide chromatography. Thirty peptides have been chromatographed for the evaluation of the proposed method, and the correlation coefficients between the calculated and the experimental retention volumes have been found to be 0.95.     

Prediction of retention volumes and UV spectra of peptides in reversed phase HPLC

A method for calculating retention volumes of linear peptides with known primary structures and the values of their UV absorption at chosen wavelengths in reversed phase HPLC are described. These parameters are calculated for every peptide on the basis of the contributions of its amino acid residues determining its degree of retention and its UV spectrum. The contribution values are experimentally found from chromatograms of the free amino acids obtained by multiwavelength photometric detection under the conditions of the peptide chromatography. Thirty peptides have been chromatographed for the evaluation of the proposed method, and the correlation coefficients between the calculated and the experimental retention volumes have been found to be 0.95.     

Determination of absorption coefficients of purified proteins by conventional ultraviolet spectrophotometry and chromatography combined with multiwavelength detection

The direct, ultraviolet spectrophotometric determination of protein absorption coefficients was found to be more reproducible and accurate when diluting was replaced by chromatography and multiwavelength detection. Four different ultraviolet spectrophotometric methods, described in the literature, were compared by calculating A0.1%280 values from the spectra of 25 proteins, obtained by chromatography. Only two methods, i.e., one based on the absorbance at 210 nm and the other on the absorbance at 205 nm, corrected for the absorbance of aromatic amino acids at that wavelength, were sufficiently accurate to be of potential use for the determination of unknown proteins. It was found, however that with uncorrected A203 values even better results could be achieved. Using 7 well-defined proteins the equation A0.1%280 = 38.69 X A280/A203 - 0.01 was established by linear regression. A0.1%280 values for 14 pure proteins calculated with this equation showed a mean deviation of only 4% from literature values. Since similar deviations were seen with 5 chromophoric and 7 glycoproteins, 3 and 7% respectively, the method may have universal applicability. In the configuration used, only 40 micrograms of a protein is required for the chromatographic determination of its absorption coefficient.     

Upper limb muscle volumes in adult subjects

Muscle force-generating properties are often derived from cadaveric studies of muscle architecture. While the relative sizes of muscles at a single upper limb joint have been established in cadaveric specimens, the relative sizes of muscles across upper limb joints in living subjects remain unclear. We used magnetic resonance imaging to measure the volumes of the 32 upper limb muscles crossing the glenohumeral joint, elbow, forearm, and wrist in 10 young, healthy subjects, ranging from a 20th percentile female to a 97th percentile male, based on height. We measured the volume and volume fraction of these muscles. Muscles crossing the shoulder, elbow, and wrist comprised 52.5, 31.4, and 16.0% of the total muscle volume, respectively. The deltoid had the largest volume fraction (15.2%+/-1%) and the extensor indicis propius had the smallest (0.2%+/-0.05%). We determined that the distribution of muscle volume in the upper limb is highly conserved across these subjects with a three-fold variation in total muscle volumes (1427-4426cm(3)). When we predicted the volume of an individual muscle from the mean volume fraction, on average 85% of the variation among subjects was accounted for (average p=0.0008). This study provides normative data that forms the basis for investigating muscle volumes in other populations, and for scaling computer models to more accurately represent the muscle volume of a specific individual.     

Prediction of peptide retention volumes in gradient reversed phase HPLC

A method of computation of retention volumes of linear peptides of known composition that contain no more than 25 amino acids in gradient reversed phase HPLC was developed. Tha method is suitable for various acetonitrile gradient profiles. The calculations were carried out on the basis of a statistical model, the parameters of which were experimental dependences of the retention of individual amino acids on acetonitrile concentration. The method developed was used to predict the chromatographic behavior of 34 peptides in four different acetonitrile gradients. The correlation coefficients between the predicted and experimental retention volumes were more than 0.9, and the average relative error of prediction was less than 15%.     

Deoxyribonueleate solutions: Sedimentation in a density gradient,partial specific volumes,density and refractive index increments,and preferential interactions

Density increments (?ρ/?c₂)°_μ in solutions of NaDNA in NaCl and CsDNA in CsCl were determined over a wide range of salt concentrations; calf thymus DNA, fragmented by sonic irradiation to a molecular weight of 4–6 × 10⁵ was used. The partial specific volume v?₂° of NaDNA at 25°C was found to ho 0.500 ml/g in water, and that of CsDNA 0.440 ml/g. Both values increase with increasing NaCl and CsCl concentration. Refractive index increments under various experimental conditions were also determined. The relevance of the density increments (at constant, chemical potential of diffusible solutes) to equilibrium sedimentation in a density gradient and the evaluation of molecular weights is discussed. Distribution coefficients of diffusible components, sometimes referred to as preferential solvation or net hydration, were derived from the density increments and partial volumes and compared with direct experimental results, whenever available, from membrane distribution and isopiestic distillation. The thermo-dynamic significance of the distribution coefficients as well as possible interpretations in terms of specific molecular mechanisms are considered.     

A method for the determination of diffusion coefficients for small molecules in aqueous solution

A method is described for determining the diffusion coefficients of small solutes in limited volumes (approximately equal to 4-9 ml) of fluid. Diffusion is measured in a three-chamber diffusion cell across a central unstirred compartment. Compartments are separated by nitrocellulose membranes. The instantaneous concentration gradient and the instantaneous flux of solute into the dilute end compartment are derived from changes in the concentration of solute in the two stirred end compartments through time. The diffusion coefficient is calculated from the slope of the least-squares regression line relating the magnitude of the instantaneous solute flux to that of the instantaneous concentration gradient. The apparatus is calibrated with a solute of known diffusivity (KCl). Diffusion coefficients thus determined in water at 25 degrees C for CaCl2 (7.54 X 10(-6) cm2.s-1), Na2-ATP (7.01 X 10(-6) cm2.s-1), 2-deoxyglucose (5.31 X 10(-6) cm2.s-1), and D-Na-lactate (5.62 X 10(-6) cm2.s-1) differed by an average of 3.7% from literature values. The method described results in accurate estimates of diffusion coefficients by a simple and relatively rapid procedure.     

Determination of concentration and aggregate size in influenza virus preparations using the true UV-absorption spectra

It is well-known that influenza virus (IV) preparations are characterized by very large contribution of light-scattering to their UV absorption spectra. With the help of so called extrapolation method we managed to measure true absorption spectra of IV preparations and to determine absorption coefficients (E0.1(1) (cm, 280)) for the intact IV virions and for IV subviral particles. These coefficients turned out to equal 1.26 +/- 0.17 and 0.96 +/- 0.11 for the virions and subviral particles respectively. The knowledge of exact IV concentration is necessary for quantitative physico-chemical studies of IV virions and their components. It is also shown that UV absorption spectra measurements allow to register IV virion aggregation. Aggregation properties of IV subviral particles were also studied.     

Left ventricular volumes and ejection fraction derived from apical two-dimensional echocardiography

Two-dimensional echocardiographic data in orthogonal apical projections were used to calculate left ventricular ejection fraction and volumes in 18 patients, 10 of whom had asynergy. The left ventricular chamber was modeled as a stack of 20 elliptical discs in order to minimize errors associated with assumptions of regular geometry. Calculations were compared to data from biplane angiography and yielded correlation coefficients of 0.91 for ejection fraction and 0.90 for volumes. The technique significantly underestimated volumes; the average ventricular volume was 161 +/- 23 ml from cineangiography and 104 +/- 25 ml from echocardiography (p < 0.001). Since this technique utilizes the most readily obtained echocardiographic views and allows for variations in ventricular architecture, its potential utility in long-term, serial evaluation of cardiac function appears promising.     

Prediction of peptide retention volumes in the gradient reversed phase HPLC

A method of computation of retention volumes of linear peptides of known composition that contain no more than 25 amino acids in gradient reversed phase HPLC was developed. The method is suitable for various acetonitrile gradient profiles. The calculations were carried out on the basis of a statistical model, the parameters of which were experimental dependences of the retention of individual amino acids on acetonitrile concentration. The method developed was used to predict the chromatographic behavior of 34 peptides in four different acetonitrile gradients. The correlation coefficients between the predicted and experimental retention volumes were more than 0.9, and the average relative error of prediction was less than 15%. The English version of the paper: Russiatn .lournal of Bioorganic Chemistry, 2008, vol. 34, no. 2; see also http://www.maik.ru.     

A critical reappraisal of Waddell's technique for ultraviolet spectrophotometric protein estimation

Waddell's method of estimating protein concentration by the difference between spectrophotometric absorptions (215-225 nm) has been reexamined. Over limited ranges of total protein, a linear relation was found for ten purified proteins; the narrowest range was between 5 and 25 micrograms/ml. Using published extinction coefficients at 280 nm for these ten proteins, protein concentration at 280 nm correlated closely with the 215 nm/225 nm difference measurements (mean difference of 2.6%). Waddell's method also accurately determined the total protein in a mixture of proteins with widely varying individual 280-nm extinction coefficients. Biuret estimates of total protein in plasma or serum gave poor correlation with measurements by Waddell's method. Within protein concentration limits, Waddell's method was linear, narrow, and more variable, both for individual proteins and for protein mixtures, than previously reported. Within these limits, the method is probably as accurate a measure of total protein as measurement by nitrogen analysis, with the advantage of being nondestructive.     

Measurement of a glycoprotein with blood group A activity by light scattering

A light scattering method for quantifying glycoproteins is presented. Conditions of ethanol concentration, wavelength, time of reading, and sample and reagent volumes have been examined. The definitive assay involves addition of 9 vol absolute ethanol to 1 vol sample, mixing, and readings at 105 degrees angle at the interval between 10 and 13 min after placing the cell into the photometer. The obtained values are temperature-dependent. Thus, temperature control is essential. In this assay, a concentration range of 10-60 micrograms glycoprotein has been used. As described, the method is independent of the chemical composition of the different glycoproteins and is barely influenced by size and shape of glycoproteins.     

Correlation between oral drug absorption in humans and apparent drug permeability coefficients in human intestinal epithelial (Caco-2) cells.

Monolayers of a well differentiated human intestinal epithelial cell line, Caco-2, were used as a model to study passive drug absorption across the intestinal epithelium. Absorption rate constants (expressed as apparent permeability coefficients) were determined for 20 drugs and peptides with different structural properties. The permeability coefficients ranged from approximately 5 x 10(-8) to 5 x 10(-5) cm/s. A good correlation was obtained between data on oral absorption in humans and the results in the Caco-2 model. Drugs that are completely absorbed in humans had permeability coefficients greater than 1 x 10(-6) cm/s. Drugs that are absorbed to greater than 1% but less than 100% had permeability coefficients of 0.1-1.0 x 10(-6) cm/s while drugs and peptides that are absorbed to less than 1% had permeability coefficients of less than or equal to 1 x 10(-7) cm/s. The results indicate that Caco-2 monolayers can be used as a model for studies on intestinal drug absorption.     

Activation volumes for intramolecular electron transfer in Escherichia coli cytochrome bo(3)

In this report we describe the activation volumes associated with the heme-heme electron transfer (ET) and CO rebinding to the binuclear center subsequent to photolysis of the CO-mixed-valence derivative of Escherichia coli cytochrome bo(3) (Cbo). The activation volumes associated with the heme-heme ET (k=1.2 x 10(5) s(-1)), and CO rebinding (k=57 s(-1)) are found to be +27.4 ml/mol and -2.6 ml/mol, respectively. The activation volume associated with the rebinding of CO is consistent with previous Cu X-ray absorption studies of Cbo where a structural change was observed at the Cu(B) site (loss of a histidine ligand) due to a change in the redox state of the binuclear center. In addition, the volume of activation for the heme-heme ET was found to be quite distinct from the activation volumes obtained for heme-heme ET in bovine heart Cytochrome c oxidase. Differences in mechanisms/pathways for heme b/heme o(3) and heme a/heme a(3) ET are suggested based on the associated activation volumes and previously obtained Marcus parameters.     

A spin label method for measuring internal volumes in liposomes or cells, applied to Ca-dependent fusion of negatively charged vesicles

A new spin label - broadening agent system for measuring trapped volumes of vesicles or cells is described. The method seems to be more advantageous than existing procedures when volumes of highly negatively charged vesicles are to be determined. The membrane permeable spin label is TEMPONE (2,2,6,6-tetramethyl piperidone-N-oxyl), and the nonpermeable broadening agent is chromium oxalate (K3Cr(C2O4)3). Absolute values for the trapped volumes down to 0.1% in 0.1 ml can be measured with an accuracy of about +/- (1-10%). The method is used to study the final volume of fused phosphatidylserine vesicles as a function of the temperature at which the Ca-induced fusion takes place.     

The partial molar volumes of anesthetics in lipid bilayers

The excess volumes of mixing of benzyl alcohol, halothane, and methoxyflurane in water and in suspensions of several lipid bilayers have been determined at 25 degrees C using a novel excess volume dilatometer. The excess volumes of mixing in water were all found to be negative, whereas in lipid suspensions they were all more positive than those in water alone. From known partition coefficients the partial molar volumes of these three solutes in the lipid bilayers were calculated. These values were all close to the molar volumes of the pure anesthetics, as was a value determined for halothane in olive oil. Halothane was studied in dipalmitoylphosphatidylcholine below its phase transition, and was found to exhibit a much larger excess volume than in any other system we studied. The potency of these three anesthetics was determined in tadpoles. It was calculated that at equi-anesthetic doses these three agents caused an expansion in egg lecithin/cholesterol (2:1) bilayers of 0.21 +/- 0.015%. This result is consistent with the hypothesis that general anesthetics act by expanding membranes.     

Pro-(carboxypeptidases A) from whole pig pancreas. Their mass, size, shape and solvation.

Sedimentation analysis and light-scattering measurements were made with the two forms of pig pancreas pro-(carboxypeptidase A), in order to determine some of their physical properties. The following values were found (the first value applies to the binary complex and the second one to the monomer). The A 1%/280.1 cm values were 19.9 +/- 0.3 and 16.3 +/- 0.3. The partial specific volumes v -0 were 0.707 +/- 0.016 cm3/g and 0.714 +/- 0.015 cm3/g. The sedimentation coefficients S 0/20,w were 4.90 +/- 0.15S and 3.75 +/- 0.15 S. The diffusion coefficients D 0/20,w were (5.8 +/- 0.1) X 10(-7) cm2/s and (6.95 +/- 0.15) X 10(-7) cm2/s. From these data the following values were calculated. Relative molecular masses Mr were 71 000 +/- 4000 and 46 000 +/- 3000. The frictional ratios f/fmin. were 1.37 +/- 0.06 and 1.31 +/- 0.07; assuming a value for the solvation of the molecules (delta = 0.5 g/g) the asymmetry values range from 3 to 5 for the binary complex and from 2 to 4 for the monomer. The Mr values found in the present work coincide with those found by means of polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate [Martínez, Avilés, SanSegundo & Cuchillo (1981) Biochem. J. 197, 141-147]. Therefore the low values obtained by those authors when using gel-filtration chromatography must be the result of the interaction of the zymogens with the gel matrix, as the asymmetry is too small to justify the large discrepancies found.     

Effect of different pion stopping volumes on the radiation response of mouse small intestine

This radiobiological investigation was based on measurements of crypt cell survival in mouse small intestine when the animals were exposed to 5 cm3 (spot), 350 cm3 and 3010 cm3 (the latter two were spot scans) pion stopping volumes generated by the multi-channel Piotron of the Swiss Institute for Nuclear Research. The experimental data obtained indicate that there was a decrease in biological effectiveness when the pion treatment volume was enlarged, irrespective of whether the pion dose was delivered in a single exposure or in four fractions. The r.b.e. values for pion beams relative to 200 kVp X-rays for the different experimental conditions used in this study are presented. The phenomenon of decreasing biological effectiveness with increasing pion stopping volume may be attributed to the following two factors: (1) when the pion stopping volume is increased there is a corresponding dilution of the high l.e.t. component of the beam; (2) the biological test system used may be sensitive to radiation dose rate which varied by a ratio of about 20 for the pion volumes used in this study.     

Development of biological tissue-equivalent phantoms for optical imaging

Optical characteristics of freshly isolated tissues depend on their color and composition. The surface backscattered profile, which account for the tissue compositional variation in fresh excised sheep's heart, lungs, bone and muscle, were measured by multi-probe reflectometer. Optical phantoms were prepared from paraffin wax by mixing a specific combination of wax color materials till the surface backscattered profile of these matched with that of the biological tissues. The optical parameters absorption coefficient (micro(a)), reduced scattering coefficient (micro(s)) and anisotropy factor (g) of these phantoms, are the same as that of biological tissues and are obtained by matching their surface backscattered profiles with that as simulated by Monte Carlo procedure. The maximum and minimum values of absorption coefficient are for the phantoms of lungs (1.0 cm(-1)) and muscle (0.02 cm(-1)), whereas, for scattering coefficient these values are for muscle (21.2 cm(-1)) and bone (13.08 cm(-1)).