Off-line Size Exclusion Chromatography Research Articles

Nanostructure development during peroxidase catalysed cross-linking of α-lactalbumin

Whereas extensive work has been done on the food functional and chemical aspects of enzymatic protein cross-linking, relatively little is known about the nanostructure and physical-chemical properties of enzymatically cross-linked protein. We investigate how nanostructure develops during enzymatic cross-linking of the 4 tyrosine residues of the globular protein apo α-lactalbumin. Protein cross-linking is catalysed by Horseradish Peroxidase, under the periodic addition of peroxide. We use on-line static and dynamic light scattering, combined with on-line UV-spectroscopy to simultaneously probe the development of nanostructure, the extent of dityrosine formation, and the catalytic state of the enzyme, as a function of the number of peroxide additions. It is found that initially, the rate of dityrosine formation is high, whereas the increase in the solution size of the cross-linked protein is limited. At later stages, the increase in solution size is significant whereas dityrosine formation slows down. Finally, the reaction stops due to enzyme inactivation. Off-line size exclusion chromatography shows that the initial phase corresponds to a fast cross-linking of monomers into small oligomers, followed by a slower joining of oligomers into large protein polymers. Consistent with this, Atomic Force Microscopy shows very heterogeneous polymers, apparently consisting of subunits that we identify with the oligomers formed in the first phase of the reaction. The dependence of the solution size on the molar mass of the cross-linked protein is determined using static and dynamic light scattering on fractionated reaction products. For sizes ranging from 30 nm to 80 nm, the protein polymers consist of 100–1000 α-lactalbumin subunits, and have molar masses of 106–107 g/mol. Apparent internal protein densities of the protein polymers calculated from these numbers are only a few weight percent, indicating a very dilute, open architecture of the cross-linked protein.

Chromatographic examination of the chemical composition and sequence distribution of copolymers from ethyl and benzyl diazoacetate

Polymers, especially copolymers, are highly complex samples and, therefore, require various setups for their thorough characterization. In this work, one- and two-dimensional chromatographic approaches were applied to characterize two homopolymers and two dissimilar copolymers prepared by rhodium-mediated carbene polymerization using ethyl and benzyl diazoacetate as the monomers: poly-(EA-ran-BnA) and poly((EA)b-(BnA-ran-EA)b). Different strategies of synthesis suggested that poly-(EA-ran-BnA) was an approximately 1:1 random copolymer and that poly((EA)b-(BnA-ran-EA)b) was a block-type copolymer of which the (BnA-ran-EA)b block is dominated by BnA. The hydrodynamic volume of the investigated polymers turned out to be comparable, i.e. size exclusion chromatography (SEC) did not separate them. Temperature-gradient interaction chromatography was not feasible and one-dimensional (solvent-)gradient-elution liquid chromatography (GELC) suffered from coelution problems. Pyrolysis-gas-chromatography (Py-GC–MS) experiments allowed determining the monomer ratio. SEC with UV and refractive-index detection indicated the presence of molecular-weight-dependent chemical heterogeneity for the poly((EA)b-(BnA-ran-EA)b) copolymer. This finding was confirmed with off-line SEC//Py-GC–MS. Only a comprehensive two-dimensional GELC×SEC setup enabled the separation of high-molecular-weight material of the less-retained homopolymer from low-molecular-weight copolymeric material. In this way it was possible to detect some high-molecular-weight homopolymeric material in one of the copolymers.

Analytical methodology for multidimensional size/branch-length distributions for branched glucose polymers using off-line 2-dimensional size-exclusion chromatography and enzymatic treatment

Instrumental and procedural optimizations are developed for a new method to obtain 2-dimensional distributions for branched homopolymers based on size and branching. The method uses 2-dimensional off-line size-exclusion chromatography (SEC) and chemical debranching, in this case using debranching enzymes on branched glucose polymers. This treatment, first presented for the 2D weight and number distributions of starch [F. Vilaplana, R.G. Gilbert, Macromolecules 43 (2010) 7321] is applied here to give 2D weight distributions as functions of size and branch length for glycogen, amylose, and native starch containing both amylopectin and amylose. Completely dissolved polysaccharides are first fractionated by size (hydrodynamic volume) using preparative SEC; the collected fractions are then debranched by addition of isoamylase, and the size distributions of the corresponding branches are analyzed for each fraction using analytical SEC with differential refractive index and with multiple-angle laser light scattering detection. Operational parameters have been optimized to provide sufficient separation resolution for each dimension (size of the whole branched macromolecule and size of the resulting branches after debranching) and to minimize degradation. These 2-dimensional distributions bring out new features in the structure of these branched polysaccharides, and offer a useful tool to elucidate relations between biosynthesis, structure, and properties.

Identification and quantification of polycarboxylates in detergent products using off-line size exclusion chromatography–nuclear magnetic resonance

The performance of many contemporary detergent products critically depends on polymers. Water-soluble polycarboxylates represent an important class of detergent polymers, and their quantitative assessment in detergent matrices stands as a considerable challenge. The presence of high levels of surfactants is a major complication, due to the strong tendency of surfactants to form micelles and to interact with the polymers. First, we addressed critical steps in the subsequent combined use of liquid extraction and off-line size exclusion chromatography–nuclear magnetic resonance (SEC–NMR) for identification and quantification of polycarboxylates in detergent products. Next, the different steps in the off-line SEC–NMR procedure were optimized with respect to precision and accuracy. This resulted in recoveries of more than 80% for maleic acid/acrylic acid copolymers; in detergent products a proportional bias of 30% is achieved. The method showed good precision with a relative standard deviation of within-laboratory reproducibility between 5% and 14%.

Mass spectrometric studies of potent inhibitors of farnesyl protein transferase—detection of pentameric noncovalent complexes

Farnesyl protein transferase (FPT) inhibition is an interesting and promising approach to noncytotoxic anticancer therapy. Research in this area has resulted in several orally active compounds that are in clinical trials. Electrospray ionization (ESI) time-of-flight mass spectrometry (TOF-MS) was used for the direct detection of a 95 182 Da pentameric noncovalent complex of alpha/beta subunits of FPT containing Zn, farnesyl pyrophosphate (FPP) and SCH 66336, a compound currently undergoing phase III clinical trials as an anticancer agent. It was noted that the desalting of protein samples was an important factor in the detection of the complex. This study demonstrated that the presence of FPP in the system was necessary for the detection of the FPT-inhibitor complex. No pentameric complex was detected in the spectrum when the experiment was carried out in the absence of the FPP. An indirect approach was also applied to confirm the noncovalent binding of SCH 66336 to FPT by the use of an off-line size exclusion chromatography followed by liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) for the detection of the inhibitor.

Matrix-assisted laser desorption/ionisation time-of-flight characterisation of biodegradable aliphatic copolyesters

Matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) and the off-line size exclusion chromatography matrix-assisted laser desorption/ionisation (SEC/MALDI) method has been applied to the structural characterisation and the molar mass (MM) determination of a series of biodegradable copolyesters synthesised by high temperature melt polycondensation reaction, and of two commercial copolyesters with the trade name Bionolle. The MALDI-TOF spectra of these copolymers showed the presence of cyclic oligomers in the lower mass region, in accord with expectations from polycondensation kinetics, and the presence of all linear species expected from their method of synthesis. The presence of unexpected linear species with olefin and carboxyl as end groups suggested the occurrence of undesirable thermal degradation processes during the melt polycondensation reaction. The absolute average molar masses obtained by the SEC-MALDI method turned out to be lower, by a factor of about two for succinate/adipate copolymers, and by a factor of three for succinate/sebacate copolymers, with respect to those computed by using polystyrene standards in SEC. Furthermore, the MALDI-TOF spectra of SEC fractions allowed not only the detection of linear and cyclic oligomers contained in these samples, but also the simultaneous determination of the average molar mass of both cyclic and linear oligomers. Due to the smaller hydrodynamic volume of cyclic chains with respect to linear ones, the ratio (M( cycle)/M( linear))( Ve) at a fixed elution volume was found to be 1.25, in good agreement with the theoretical value of 1.24. Copyright 2000 John Wiley & Sons, Ltd.

Direct sample fraction deposition using electrospray in narrow-bore size-exclusion chromatography/matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for polymer characterization

A direct sample fraction deposition method was developed for off-line size-exclusion chromatography (SEC)/matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry. By using electrospray, the SEC eluent, together with a suitable matrix solution added coaxially, was directly deposited on the MALDI plate. Owing to the formation of very small droplets in electrospray, solvent evaporation is much faster. The fractionation volume in narrow-bore SEC, which can directly be collected in one MALDI spot, can easily be optimized in the range of a few microlitres. In addition, fairly homogeneous sample spots were obtained. The possible influence of composition variation of the SEC effluent on the analytical results using direct fraction deposition was investigated; no substantial effects were observed. The applicability of the method was demonstrated by characterizing a broad poly(methyl methacrylate) sample. Copyright 2000 John Wiley & Sons, Ltd.