Molecular weight estimation using sodium dodecyl sulfate-pore gradient electrophoresis

Pore gradient electrophoresis (PGE) in the presence of sodium dodecyl sulfate (SDS) provides a means for high resolution fractionation of multicomponent protein systems and permits estimation of molecular weights for macromolecules ranging from 10³ to 10⁶. We have evaluated the performance of several methods used to construct calibration curves for estimation of molecular weights using SDS-PGE. A linear relationship between the logarithm of molecular weight, log (M_r), and the logarithm of the relative mobility, log (R_l), can be obtained for a 30-fold range of molecular weights. However, this range of linearity depends on the choice of the concentration gradient, the degree of crosslinking of the gel, and on the nature of the underlying relationship between the retardation coefficient, K_R, and the molecular weight. An empirical relationship, first introduced by Lambin et al. (1976, Anal. Biochem.74, 567) between log (M_r) and the logarithm of the gel concentration at the position reached by the protein, log (%T), provides better linearity over a wider molecular weight range than does the use of log (R_l). We have compared these relatienships by experimental analysis of 10 standard proteins and by a theoretical analysis of an idealized model system. A computer program has been developed which provides appropriate statistical estimation of the molecular weight for an unknown protein, together with its standard error and 95% confidence limits. A new method has also been developed for analysis of nonlinear calibration curves in terms of molecular weight versus distance migrated, based on a theoretically justifiable, physical-chemical model. This model implies that either the relationship between log (M_r) and log (R_l) or the one between log (M_r) and log (%T) will become nonlinear as the range of molecular weight is extended. We suggest that the use of a nonlinear least-squares curve-fitting procedure provides an optimal method for molecular weight estimation when sufficient data are available. Based on these findings, a general strategy is presented for estimation of molecular weights by polyacrylamide gel electrophoresis.     

Murine malaria. I. Measurement of the mean rate of increase in vivo of Plasmodium berghei

Swiss mice were inoculated intraperitoneally with RBC infected with Plasmodium berghei. The moment a certain parasitemia was reached in each individual mouse was estimated by means of linear interpolation. The relationship between latent period and log inoculum was investigated by means of simple linear regression. The slopes of the latent period per log inoculum curves were significantly different using different donor mice inoculated with serial 10-fold dilution of infected RBC and exsanguinated in the same phase of the infection. It could not be demonstrated that the slopes of the regression lines depended on the stage of the disease of the donor mouse.     

Precision of sodium dodecyl sulfate-polyacrylamide-gel electrophoresis for the molecular weight estimation of a membrane glycoprotein: studies on bovine rhodopsin.

Criteria for assessing the precision and accuracy of methods for estimation of molecular weight for proteins using sodium dodecyl sulfate-polyacrylamide-gel electrophoresis have been applied to rhodopsin from bovine visual cell outer segment membranes. Various methods of preparing this hydrophobic protein for electrophoresis differ in their ability to solubilize and disaggregate polypeptide constituents of the outer segment membrane, with resultant variations in the pattern of protein bands and the apparent molecular weight of rhodopsin. Even with optimal solubilization and disaggregation, the behavior of rhodopsin relative to a series of standard proteins is such that the apparent molecular weight decreases systematically from 40,400 to 34,500 as the acrylamide concentration increases from 4 to 10%. As demonstrated by Ferguson plots of logR_f vs gel concentration and split gel experiments, this discrepancy is explained by the fact that the extrapolated R_f for zero gel concentration (Y₀) for rhodopsin is significantly lower than the Y₀'s for the soluble proteins used as molecular weight standards. In such cases, a possibly more reliable molecular weight estimate is obtained by plotting the retardation coefficient (K_R) vs molecular weight. This method yields a value of 29,500 ± 1000 for bovine rhodopsin if only the errors in measurement of R_f are considered and a quadratic relationship between K_R and molecular weight is used. Using weighted linear regression for K_R vs molecular weight, we obtain a molecular weight estimate of 32,700 ± 5000 when the uncertainty in the calibration curve is considered. Because of uncertainties regarding the detergent-binding properties of rhodopsin and the relationship of its Stokes radius to its molecular weight by comparison with the soluble protein standards, these values must be viewed with caution.     

Reversed-phase thin-layer chromatography in the parametrization of lipophilicity of some series of arylaliphatic acids

In the series of arylacetic acid and β-aryl-n-butyric acids, chromatography was carried out on a thin layer of silica gel impregnated with silicone oil, with 50% acetone as mobile phase. A separation mechanism in this system was evaluated using relationships between R_M values and the concentration of the lipophilic solvent in the silica-gel layer. It was found, that both partition and adsorption mechanisms participate, and that the adsorption effect increases with decreasing lipophilicity of the acids. The dichotomy of the mechanism manifests itself in the non-linear course of the relationships between R_M values and π parameters, or fragmental constants f derived from the partition system n-octanol—water. Such relationships can be expressed by a single quadratic dependence between lipophilic parameters and R_M values, or by two separate linear expressions with different slopes for different regions of substituent lipophilicities. The linear dependence between R_M and π at the lower range of lipophilicity is most probably made possible by significant linear dependence between π parameters and molecular surface areas of the substituents.     

A Proteomic Approach to Understand the Role of the Outer Membrane Porins in the Organic Solvent-Tolerance of Pseudomonas aeruginosa PseA

Solvent-tolerant microbes have the unique ability to thrive in presence of organic solvents. The present study describes the effect of increasing hydrophobicity (log P_ow values) of organic solvents on the outer membrane proteome of the solvent-tolerant Pseudomonas aeruginosa PseA cells. The cells were grown in a medium containing 33% (v/v) alkanes of increasing log P_ow values. The outer membrane proteins were extracted by alkaline extraction from the late log phase cells and changes in the protein expression were studied by 2-D gel electrophoresis. Seven protein spots showed significant differential expression in the solvent exposed cells. The tryptic digest of the differentially regulated proteins were identified by LC-ESI MS/MS. The identity of these proteins matched with porins OprD, OprE, OprF, OprH, Opr86, LPS assembly protein and A-type flagellin. The reported pI values of these proteins were in the range of 4.94–8.67 and the molecular weights were in the range of 19.5–104.5 kDa. The results suggest significant down-regulation of the A-type flagellin, OprF and OprD and up-regulation of OprE, OprH, Opr86 and LPS assembly protein in presence of organic solvents. OprF and OprD are implicated in antibiotic uptake and outer membrane stability, whereas A-type flagellin confers motility and chemotaxis. Up-regulated OprE is an anaerobically-induced porin while Opr86 is responsible for transport of small molecules and assembly of the outer membrane proteins. Differential regulation of the above porins clearly indicates their role in adaptation to solvent exposure.     

Reliability of molecular weight determination of proteins by polyacrylamide gradient gel electrophoresis in the presence of sodium dodecyl sulfate

Thirty-four proteins with molecular weights (MWs) between 1.3 × 10⁴ and 9.5 × 10⁵ and seven polypeptide chains of some of these proteins were incubated in sodium dodecyl sulfate and submitted to electrophoresis in a polyacrylamide gradient gel (3 to 30%; relative percentage of bisacrylamide: 8.4%). Analysis of the results confirmed the reliability of the relationship: log(MW) = alog(T) + b, where T is the polyacrylamide concentration reached by a protein, a is the slope, and b is the Y intercept of the regression line. In fact the mean deviation between the expected and the observed MW was ±5.5%. In addition we have shown that MWs of unreduced and reduced proteins can be estimated with the same accuracy and tha MWs of glycoproteins can also be determined with the same accuracy as other proteins.     

The gel-filtration behaviour of proteins related to their molecular weights over a wide range

1. Correlation between elution volume, V_e, and molecular weight was investigated for gel filtration of proteins of molecular weights ranging from 3500 (glucagon) to 820000 (α-crystallin) on Sephadex G-200 columns at pH7·5. 2. Allowing for uncertainties in the molecular weights, the results for most of the carbohydrate-free globular proteins fitted a smooth V_e–log(mol.wt.) curve. In the lower part of the molecular-weight range the results were similar to those obtained with Sephadex G-75 and G-100 gels. 3. V_e–log(mol.wt.) curves based on results with the three gels are taken to represent the behaviour of `typical' globular proteins, and are proposed as standard data for the uniform interpretation of gel-filtration experiments. 4. Some glycoproteins, including γ-globulins and fibrinogen, do not conform to the standard relationship. The effect of shape and carbohydrate content on the gel-filtration behaviour of proteins is discussed. 5. As predicted by the theoretical studies of other authors, correlation exists between the gel-filtration behaviour and diffusion coefficients of proteins. 6. The lower molecular-weight limit for complete exclusion of typical globular proteins from Sephadex G-200 varies with the swelling of the gel, but is usually >10⁶. 7. The concentration-dependent dissociation of glutamate dehydrogenase was observed in experiments with Sephadex G-200, and the sub-unit molecular weight estimated as 250000. The free sub-units readily lose enzymic activity. 8. Recognition of the atypical gel-filtration behaviour of γ-globulins necessitates an alteration to several molecular weights previously estimated with Sephadex G-100 (Andrews, 1964). New values are: yeast glucose 6-phosphate dehydrogenase, 128000; bovine intestinal alkaline phosphatase, 130000; Aerobacter aerogenes glycerol dehydrogenase, 140000; milk alkaline phosphatase, 180000.     

Rapid determination of logarithmic partition coefficients between n-octanol and water using micellar electrokinetic capillary chromatography

Micellar electrokinetic capillary chromatography (MECC) was evaluated as a rapid screening tool for the determination of logarithmic partition coefficients between n-octanol-water (logP_ow). The technique is performed by electrochromatographing a mixture of standards of known log P_ow. The logarithmic capacity factor of each standard was plotted against its log P_ow to form a linear calibration curve for a given set of chromatographic conditions. The log P_ow of an unknown is calculated by using its chromatographically determined capacity factor and extracting the log P_ow value from the calibration curve. The method was evaluated with a set of model compounds with known log P_ow. The accuracy of the method was examined and found to be within the limits required for screening purposes. The correlation of log P_ow values determined using HPLC and MECC for some novel compounds was examined. This technique allows the screening of log P_ow at a rate of four samples per hour with minimal sample requirements (<μg) and with extremely small solvent waste generated.     

Histone-like protein in the Archaebacterium Sulfolobus acidocaldarius

The Archaebacterium Thermoplasma acidophilum contains a basic chromosomal protein remarkably similar to the histones of eukaryotes. Therefore, it was of interest to examine a different Archaebacterium for similar proteins. We chose to examine Sulfolobus acidocaldarius because it is thermophilic, like T. acidophilum, but nevertheless the two organisms are not particularly closely related. Two major chromosomal proteins were found in S. acidocaldarius. The smaller of these was soluble in 0.2 M H₂SO₄ and had a molecular weight of 14500. The larger was acid-insoluble and had a molecular weight of about 36000. Together, the proteins protected about 5% of the DNA against nuclease digestion and stabilized about 50% against thermal denaturation. Overall, the properties of these proteins were intermediate between those of the Escherichia coli protein HU and T. acidophilum protein HTa.     

Synthesis of early heat shock proteins in young leaves of barley and sorghum

The in vivo synthesis of early heat-shock proteins in young leaves of barley (Hordeum vulgare L.) and sorghum (Sorghum bicolor L.) was studied by one- and two-dimensional electrophoresis. Analysis of whole leaf protein patterns demonstrated clearly the enhanced resolution of heat-shock proteins, especially those of low molecular weight, when separated by two-dimensional electrophoresis. Comparison between the two cereals showed that a greater number and diversity of heat-shock proteins were induced in the subtropical C₄ (sorghum) species compared to the temperate C₃ (barley) species. Fractionation of whole leaf proteins into soluble and membrane fractions showed the majority of heat-shock proteins to be associated with the soluble fraction in both sorghum and barley. However, several low molecular mass (17-24 kilodalton) heat-shock proteins were clearly identified in the membrane fractions, indicating a likely association with thylakoid membranes in vivo during the early stages of a heat-shock response in both species.     

Solution thermodynamic approach to analyze protein stability in aqueous solutions

Protein thermal stability was analyzed by a solution thermodynamic approach. The small energetic differences in hydrogen-bonds (HB) among amino acid resdues and water molecules were proved to be amplified by the large number of HB involved to bring about the equilibrium shift from folding to unfolding of proteins. In aqueous solutions, water activity (A_w) plays a key role in protein stability. Therefore, A_w was precisely determined for various solutions and its relationship with solution structure was discussed. Wyman-Tanford analysis based on A_w showed linear regressions, without exception, between protein unfolding-ratio and A_w for lysozyme, ribonuclease A, and α-chymotrypsinogen A in various solutions with sugars, osmolytes, alcohols, and protein denaturant. From this linear regression, the free energy difference, ΔΔG, for a protein in a solution and in pure water, was easily obtained. Protein stability in a solution was proved to be determined by a balance between hydration and solute-binding effects to the protein and also by solution structure, which indirectly affects the hydrophobic interaction in a protein molecule. Temperature dependence of HB on protein stability suggested its interrelationship with hydrophobic interaction.     

Structure-activity relationships in papain and bromelain ligand interactions

The parameters K_m and k_cat were determined for 16 methyl hippurates (CH₃OCOCH₂NHCOC₆H₄-X) hydrolyzed by papain. A simple linear relationship is found between log $\text{1}\text{K}{}_{\mathrm{m}}$ and the hydrophobic substituent constant π. It is found that log k_cat is parabolically related to π. The results with papain are compared with results obtained by Hawkins and Williams with the enzyme bromelain. The two enzymes behave in a similar fashion.     

Transgelin: An androgen-dependent protein identified in the seminal vesicles of three Saharan rodents

During the breeding season, a major androgen-dependent protein with an apparent molecular weight of 21 kDa was isolated and purified from the seminal vesicles of three Saharan rodents (MLVSP21 from Meriones libycus, MSVSP21 from Meriones shawi, and MCVSP21 from Meriones crassus). The 21-kDa protein was isolated and purified from soluble seminal vesicle proteins of homogenate by one-dimensional polyacrylamide gel electrophoresis (SDS-PAGE). Using polyclonal antibodies directed against POSVP₂₁ (Psammomys obesus seminal vesicles protein of 21 kDa), a major androgen-dependent secretory protein from sand rat seminal vesicles, identified previously as transgelin, we showed an immunological homology with POSVP₂₁ by immunoblotting. These three major androgen-dependent proteins with a same apparent molecular weight of 21 kDa designated as MLVSP₂₁ (Meriones libycus seminal vesicles protein of 21 kDa), MSVSP₂₁ (Meriones shawi seminal vesicles protein of 21 kDa), and MCVSP₂₁ (Meriones crassus seminal vesicles protein of 21 kDa) were localized by immunohistochemistry and identified by applying a proteomic approach. Our results indicated that the isolated proteins MLSVP₂₁, MSSVP₂₁, and MCSVP₂₁ seem to correspond to the same protein: the transgelin. So that transgelin can be used as a specific marker of these rodent physiological reproduction mechanisms.     

Proteomics Profiling of Human Synovial Fluid Suggests Increased Protein Interplay in Early-Osteoarthritis (OA) That Is Lost in Late-Stage OA

The underlying molecular mechanisms in osteoarthritis (OA) development are largely unknown. This study explores the proteome and the pairwise interplay of proteins in synovial fluid from patients with late-stage knee OA (arthroplasty), early knee OA (arthroscopy due to degenerative meniscal tear), and from deceased controls without knee OA. Synovial fluid samples were analyzed using state-of-the-art mass spectrometry with data-independent acquisition. The differential expression of the proteins detected was clustered and evaluated with data mining strategies and a multilevel model. Group-specific slopes of associations were estimated between expressions of each pair of identified proteins to assess the co-expression (i.e., interplay) between the proteins in each group. More proteins were increased in early-OA versus controls than late-stage OA versus controls. For most of these proteins, the fold changes between late-stage OA versus controls and early-stage OA versus controls were remarkably similar suggesting potential involvement in the OA process. Further, for the first time, this study illustrated distinct patterns in protein co-expression suggesting that the interplay between the protein machinery is increased in early-OA and lost in late-stage OA. Further efforts should focus on earlier stages of the disease than previously considered.     

The lysis of isolated fish (Oncorhynchus mykiss) gill epithelial cells by surfactants

1. The interaction of a wide range of surfactants with isolated gill epithelial cells of rainbow trout (Oncorhynchus mykiss) was investigated as a function of the surfactant concentration up to and above the critical micelle concentration (cmc). The surfactants included a homologous series of n-alkyl sulphates, single and double chain tri and dimethylammonium bromides (TABs and DABs), cholates and the nonionics n-octylglucoside and Triton X-100.2. With the exception of the C₂₂ alkyl chain TAB and the double chain [(C₁₂)₂] DAB, the surfactants solubilized between 84 and 100% of the cell protein at high concentrations (>cmc).3. At low concentrations n-dodecyltrimethylammonium bromide and, to a lesser extent, Triton X-100 and sodium n-dodecylsulphate release a larger proportion of cell protein than they solubilize lipid in contrast to sodium cholate which initially preferentially solubilizes cell lipid. This differential pattern of solubilization is similar to that observed for other plasma membranes such as those of human erythrocytes and platelets.4. The surfactant concentration required to solubilize 50% (S_50%) of cell protein increases with the cmc. There is an approximately linear relationship between log(S_50%) and log cmc.5. Light microscopy shows that the surfactants at high concentrations (>cmc) fragment the secondary lamellae of the gill filaments.     

Conformation of mucous glycoproteins in aqueous solvents

Light-scattering techniques have been used to measure the z-average radius of gyration R_g z-average translational diffusion coefficient D_t and weight–average molecular weight M_w of porcine submaxillary mucin (PSM) in solution. PSM isolated at low shear in the presence of protease inhibitors has a M_w about twice as large as a sample prepared without these precautions. The former sample has a M_w of 17 × 10⁶ in 0.1M NaCl, which decreases to 8 × 10⁶ in 6M guanidine hydrochloride (GdnHCl) and then to 2 × 10⁶ on addition of 0.1M mercaptoethanol to the 6M GdnHCl solution. The R_g or D values obtained for PSM in this work superimpose with those of other authors for different mucin glycoproteins, leading to linear log–log relationships to the molecular weight of the protein core. Comparison of these results with those in the literature for denatured proteins suggest that mucins are linear random coils in which the protein core is stiffened by the presence of the oligosaccharide side chains. The length of the oligosaccharides and the nature of the solvent have little effect on the extension of the protein core. This suggests that the stiffness of the protein core is maintained by steric repulsion of the residues at the beginning of the oligosaccharide chains.     

Self-association of oxyhaemoglobin. A nuclear magnetic relaxation study in H2O/D2O solutions

The proton and deuterium longitudinal relaxation rates were Studied at room temperature up to the highest protein concentrations in oxyhaemoglobin solutions of different H₂O/D₂O composition. The deuterium relaxation rates followed the experimentally well known single linear dependence on protein concentration, the slopes being little influenced by solvent (D₂O/H₂O) composition. The proton ralaxation rates show two different liner dependences on haemoglobin concentration. The entire concentration range is described by two straight lines with the threshold concentration about 11 mM (in haem), The ratio of the slopes is 1.6 (high-to-low Hb-conc.). Only in the higher concentration range two T₁'s were observed if the solvent contained more than half of D₂O. The slow relaxation phase of protons has T₁'s similar to those measured in solutions with less than half of D₂O. The relaxation of the other phase was ten times faster. The ratio of the proton populations in these two phases was equal to 2 (slow-to-fast) and independent of protein concentration. The fast relaxing protons are attributed to water molecules encaged within two or more haemoglobin molecules which associate for times long enough on the PMR time-scale.     

Heme-linked ionization and chloride binding in intestinal peroxidase and lactoperoxidase.

Hog intestinal peroxidase and bovine lactoperoxidase exhibited similar spectral shifts upon pH alteration. From spectrophotometric titrations, it was found that there are hemelinked ionizations of pK_a = 4.75 in intestinal peroxidase and pK_a = 3.5 in lactoperoxidase. The apparent pK_a (pK_a′) increased with the increase in chloride concentration. The pK_a′ vs log[Cl^?] plots showed that the chloride forms complex with the acid forms of these enzymes with a dissociation constant (pK = 2.7). Although the dissociation constant (K_d) of the peroxidase-cyanide complexes is nearly independent of pH, cyanide competed with chloride in the acidic pH region. The slopes of logK_d vs log[Cl^?] were 1.0 for intestinal peroxidase and 0.5 for lactoperoxidase. The reaction of hydrogen peroxide with these peroxidases was also affected by chloride, similarly as the reaction with cyanide was. The results were explained by assuming that protonation occurs at the distal base and destroys the hydrogen bond between the base and a water molecule at the sixth coordinate position of the heme iron.     

The use of liquid polyacrylamide in electrophoresis: III. Properties of liquid polyacrylamide in the presence of cellulose acetate

Solutions of uncross-linked liquid polyacrylamide (LPA) stabilized by cellulose acetate are shown to represent efficient molecular sieves for RNA and for sodium dodecyl sulfate-protein complexes. Endosmosis and evaporation have to be considered to obtain corrected estimates of mobilities of the macro-ions. The efficiency of molecular sieving is strongly influenced by the chain length of the single polymer molecules. This can be shown both in terms of the steepness of the relationship between logarithm of molecular weight and mobility and in the relationship between log (K_R) and log molecular weight. Comparable chain length effects are found in cross-linked polyacrylamide, suggesting a common mechanism of action of the two electrophoretic sieving media. The correlation of polymer viscosity and of the impedance towards migration of macro-ions, as demonstrated by LPA-electrophoresis, suggests the participation of hydrodynamic interactions in the molecular sieving process.     

Molecular weights of protein multimers from polyacrylamide gel electrophoresis

A nonlinear relationship between polyacrylamide gel electrophoresis (PAGE) retardation coefficients (K_R) and molecular weights has been observed during analysis of several multimeric proteins. Although the deviation from linearity over a wide range of molecular weights is slight, it can lead to significant errors in the estimation of the sizes of individual multimers. Two alternative methods of analysis of PAGE results are compared and demonstrated to yield linear relationships for multimeric proteins having molecular weights as high as 900,000.