Quantitative Size-exclusion Chromatography Research Articles

Hsp70 Oligomerization Is Mediated by an Interaction between the Interdomain Linker and the Substrate-Binding Domain

Oligomerization in the heat shock protein (Hsp) 70 family has been extensively documented both in vitro and in vivo, although the mechanism, the identity of the specific protein regions involved and the physiological relevance of this process are still unclear. We have studied the oligomeric properties of a series of human Hsp70 variants by means of nanoelectrospray ionization mass spectrometry, optical spectroscopy and quantitative size exclusion chromatography. Our results show that Hsp70 oligomerization takes place through a specific interaction between the interdomain linker of one molecule and the substrate-binding domain of a different molecule, generating dimers and higher-order oligomers. We have found that substrate binding shifts the oligomerization equilibrium towards the accumulation of functional monomeric protein, probably by sequestering the helical lid sub-domain needed to stabilize the chaperone: substrate complex. Taken together, these findings suggest a possible role of chaperone oligomerization as a mechanism for regulating the availability of the active monomeric form of the chaperone and for the control of substrate binding and release.

ANALYSIS OF THE COMPOSITION OF COMPLICATED BINARY MIXTURE BY QUANTITATIVE SEC

The present paper provides a quantitative size exclusion chromatography(SEC) method based on the principle of absolute quantification of SEC to solve the problem.The conventional data treatment procedure for the D.R.I.signal of SEC H(V) is improved first by dividing it with the injected sample weight and leads to a novel defined weight normalized distribution H-w(V) and its integral I-w(V).These two distributions reflect the response constant of the sample in addition to the conventional relative total area normalized distribution F(V).The difference of the response constants of the mixtures provides a method to compute their composition from the H-w(V) or I-w(V) of its single component by method of summation.The calculations are carried out in a Mathcad worksheet with the principle of least sum square deviation.The method was applied to mixtures of a kind of industrial asphalt and paraffinic diluents as an example and successful results are obtained.

Analysis of composition complicated binary mixture by quantitative SEC

The analyses of the composition of a binary mixture composed of two kinds of industrial complicated materials have great importance for formulation in practice. The present paper provides a quantitative size exclusion chromatography (SEC) method based on the principle of absolute quantification of SEC to solve the problem. The conventional data treatment procedure for the differential refractive index (DRI) signal of SEC H(V) is improved first by dividing it with the injected sample weight and leads to a novel defined weight normalized distribution H w(V) and its integral I w(V). These two distributions reflect the response constant of the sample in addition to the conventional normalized distribution F(V). The difference of the average response constants of the composing components provides a sensitive method to compute the composition of their mixture from its H w(V) or I w(V). The method was applied to mixtures of a kind of industrial asphalt and paraffin diluents as an example, and successful results are obtained.

Biophysical Characterization of Structural Properties and Folding of Interleukin-1 Receptor Antagonist

Structural properties and folding of interleukin-1 receptor antagonist (IL-1ra), a therapeutically important cytokine with a symmetric β-trefoil topology, are characterized using optical spectroscopy, high-resolution NMR, and size-exclusion chromatography. Spectral contributions of two tryptophan residues, Trp17 and Trp120, present in the wild-type protein, have been determined from mutational analysis. Trp17 dominates the emission spectrum of IL-1ra, while Trp120 is quenched presumably by the nearby cysteine residues in both folded and unfolded states. The same Trp17 gives rise to two characteristic negative peaks in the aromatic CD. Urea denaturation of the wild-type protein is probed by measuring intrinsic and extrinsic (binding of 1-anilinonaphthalene-8-sulfonic acid) fluorescence, near- and far-UV CD, and 1D and 2D ( 1H– 15N heteronuclear single quantum coherence (HSQC)) NMR. Overall, the data suggest an essentially two-state equilibrium denaturation mechanism with small, but detectable structural changes within the pretransition region. The majority of the 1H– 15N HSQC cross-peaks of the folded state show only a limited chemical shift change as a function of the denaturant concentration. However, the amide cross-peak of Leu31 demonstrates a significant urea dependence that can be fitted to a two-state binding model with a dissociation constant of 0.95 ± 0.04 M. This interaction has at least a five times higher affinity than reported values for nonspecific urea binding to denatured proteins and peptides, suggesting that the structural context around Leu31 stabilizes the protein–urea interaction. A possible role of denaturant binding in inducing the pretransition changes in IL-1ra is discussed. Urea unfolding of wild-type IL-1ra is sufficiently slow to enable HPLC separation of folded and unfolded states. Quantitative size-exclusion chromatography has provided a hydrodynamic view of the kinetic denaturation process. Thermodynamic stability and unfolding kinetics of IL-1ra resemble those of structurally and evolutionary close IL-1β, suggesting similarity of their free energy landscapes.

Heterologous expression and characterization of a novel branching enzyme from the thermoalkaliphilic anaerobic bacterium Anaerobranca gottschalkii

The gene encoding the branching enzyme (BE) from the thermoalkaliphilic, anaerobic bacterium Anaerobranca gottschalkii was fused with a twin arginine translocation protein secretory-pathway-dependent signal sequence from Escherichia coli and expressed in Staphylococcus carnosus. The secreted BE was purified using hydrophobic interaction and gel filtration chromatography. The monomeric enzyme (72 kDa) shows maximal activity at 50 degrees C and pH 7.0. With amylose the BE displays high transglycosylation and extremely low hydrolytic activity. The conversion of amylose and linear dextrins was analysed by applying high-performance anion exchange chromatography and quantitative size-exclusion chromatography. Amylose (10(4)-4 x 10(7) g/mol) was converted to a major extent to products displaying molecular masses of 10(4)-4 x 10(5) g/mol, indicating that the enzyme could be applicable for the production of starch or dextrins with narrow molecular mass distributions. The majority of the transferred oligosaccharides, determined after enzymatic hydrolysis of the newly synthesized alpha-1,6 linkages, ranged between 10(3) and 10(4) g/mol, which corresponds to a degree of polymerisation (DP) of 6-60. The minimal donor chain length is DP 16. Furthermore, the obtained results support the hypotheses of a random endocleavage mechanism of BE and the occurrence of interchain branching.

Chromatographic characterization of synthetic peptides: SPf66 malaria vaccine

The development and validation of a quantitative size-exclusion chromatography (SEC) method for SPf66 malaria vaccine was achieved. The results show the reliability of the analytical method for the intended use. SPf66 malaria vaccine characterization was performed using both relative techniques such as the conventional SEC and absolute techniques: mass spectrometry and multi-angle laser-light scattering detection. The relative and absolute molecular masses were in the 4600–18 000 Da range. The results clearly indicate the presence of the monomer and dimer species, whereas the third species could be the trimer or tetramer.

Synthesis and characterization of organosiloxane modified segmented polyether polyurethanes

Segmented polyurethanes derived from a 1000 M̄n hydroxyl terminated polytetramethylene oxide soft segment, 4,4′-methylene diphenyl diisocyanate, MDI, and 1,4-butanediol were modified with a 1200 M̄n secondary aminoalkyl functional polydimethylsiloxane (PDMS) oligomer via solution polymerization in tetrahydrofuran (THF)/dimethylacetamide (DMAC). Various compositions were studied using FTIR and NMR spectroscopy, thermal analysis, quantitative size exclusion chromatography (SEC), cone calorimetry, transmission electron microscopy, XPS and mechanical testing. The results suggest that, with as little as 15% of the polydimethylsiloxane one may reduce the cone calorimetry heat release rate by a factor of about 2/3 and hence improve fire resistance, while maintaining mechanical behavior. It is suggested that the low surface energy characteristics of PDMS promote migration to the air–polymer interface to form a predominately PDMS enriched surface. The latter is oxidized at elevated temperatures in air to a silicate-like material and this serves as a protective layer, which further reduces burning of the underlying polyurethane. Dynamic mechanical behavior and electron microscopy suggest that a complex mutiphase structure is produced, particularly at low PDMS weight fractions.

Quantitative size exclusion chromatography of polypropylene II: Analysis systems

AbstractQuantitative size exclusion chromatography (SEC) was considered a system with the following components: sample preparation, fractionation, detection, calibration, and resolution correction. Four systems were evaluated: I was 3 columns with “conventional single detector interpretation”; II was 4 columns with concentration correction and detector assessment; III was concentration correction applied to the data of I; IV was two development “mixed bed” columns. Analysis of polystyrene standards included calculation of their molecular weight averages and use of the Glöckner “T” measure of resolution as well as “specific resolution index.” Systems I and III provided the best high molecular weight results. System IV allowed 12–20 min analysis times and provided a highly linear calibration curve with very good reproducibility; however, it showed significantly worse resolution at high molecular weights. Plots of molecular weight averages determined from SEC versus their known true values were particularly useful. Concentration correction using the Rudin model moved different concentration data toward a common universal calibration curve and generally lowered molecular weight averages. Narrow polystyrene standards required relatively high concentrations for precise molecular weight averages and therefore, their averages were not good indicators of the need for concentration correction. Analysis of polypropylene samples corroborated the lower high molecular weight resolution of System III. Concentration correction did significantly change the polypropylene molecular weight distribution but did not affect the result of the kinetic model fitting.