Proteins molecular sizes

Most peptides and proteins are currently formulated as parenteral formulations because of their poor oral bioavailability. Nevertheless, oral delivery of peptides and proteins would be the preferred route of administration if bioavailability issues could be overcome, as it offers the advantages of convenient, pain-free administration. Although various factors such as permeability, chemical and metabolic stability and gastrointestinal transit time can affect the rate and extent of absorption of orally administered peptides and proteins, molecular size is generally considered the ultimate obstacle [36]. 

Extraction of Unreduced Glutelins. Protein molecular sizes or size distributions often influence functional properties, necessitating extraction and characterization of native proteins. Various procedures have solubilized and fractionated unreduced wheat glutenin using dilute acetic acid [34], solutions differing in HCl concentration [35-37], or 50% and 70% I-propanol [38]. 

Another example is the purification of a P-lactam antibiotic, where process-scale reversed-phase separations began to be used around 1983 when suitable, high pressure process-scale equipment became available. A reversed-phase microparticulate (55—105 p.m particle size) C g siUca column, with a mobile phase of aqueous methanol having 0.1 Af ammonium phosphate at pH 5.3, was able to fractionate out impurities not readily removed by hquid—hquid extraction (37). Optimization of the separation resulted in recovery of product at 93% purity and 95% yield. This type of separation differs markedly from protein purification in feed concentration ( i 50 200 g/L for cefonicid vs 1 to 10 g/L for protein), molecular weight of impurities (<5000 compared to 10,000—100,000 for proteins), and throughputs ( i l-2 mg/(g stationary phasemin) compared to 0.01—0.1 mg/(gmin) for proteins). 

The selectivity of a gel, defined by the incremental increase in distribution coefficient for an incremental decrease in solute size, is related to the width of the pore size distribution of the gel. A narrow pore size distribution will typically have a separation range of one decade in solute size, which corresponds to roughly three decades in protein molecular mass (Hagel, 1988). However, the largest selectivity obtainable is the one where the solute of interest is either totally excluded (which is achieved when the solute size is of the same order as the pore size) or totally included (as for a very small solute) and the impurities differ more than a decade in size from the target solute. In this case, a gel of suitable pore size may be found and the separation carried out as a desalting step. This is very favorable from an operational point of view (see later). 

Proteins are separated on Zorbax GF columns based on their hydrodynamic size, which may be related to the proteins molecular weights (Fig. 3.10). Under ideal conditions, two proteins whose molecular sizes differ by a factor of 2 can be baseline separated. 

As with other size-exclusion techniques, the pore size of the selected Zorbax GF column should provide resolution over the molecular size range of the proteins that are to be separated. The Zorbax GF-250 column separates proteins in the range of 4000 to 400,000 Da. The Zorbax GF-450 provides separation over the range of 10,000 to 1,000,000 Da. When these two columns are coupled, they can be used to separate proteins with molecular weights of 4000 to 1,000,000. 

In theory, SEC of proteins depends only on their molecular size. Sometimes the size of a protein varies with the ionic strength of the buffer (5,6). The concentration of salt not only affects the conformation of the protein, but can also influence the chromatographic separation itself. Additional retention... 

Charged macromolecules, such as proteins or polymers, are often separated elec-trophoretically. The rate of migration through an electric field increases with net charge and field strength. Molecular size of analytes and viscosity of separation media both have inverse relationships with rate of migration. These variables must all be taken into account in order to optimize the conditions for an efficient electrophoretic separation. 

Luciferin binding protein (LBP) binds luciferin at pH 8 but not at pH 6 (Fogel and Hastings, 1971) thus, LBP inhibits the luciferin-luciferase reaction at pH 8 but not at pH 6. Luciferin bound to LBP is stable, differing from the free form of luciferin that is extremely unstable. The molecular size of the Gonyaulax LBP was considered... 

Component Molecular Size Number of Peptide Subunits Prosthetic Croups Topology in Inner Membrane Abundance in Inner Membrane (nmol per mg Protein. Data for Cardiac Mitochondria) Proton Movements (the Stoichiometry is Discussed in Appendix 3)... 

There are approximately 200 other proteins present in bone, though most of them are present only in trace amounts (Delmas et al., 1984 Linde et al., 1980, as cited in van Klinken, 1991). The second most common bone protein, osteocalcin, comprises 1-2 weight % of total fresh bone. Osteocalcin bonds with both the bone mineral fraction and bone collagen, but it seems to be unstable in solutions. Due to its small molecular size and strong mineral stabilization, osteocalcin can survive up to 50.000 years (C.l. Smith et al., 2005), and it may offer an alternative to the use of collagen in paleoenvironmental stable isotope research. However, osteocalcin s role and importance in this field of study has yet to be defined (Collins et al., 2002). 

Protein Mr Molecular size (in nm) pi %R pH value of the protein solutions... 

Coelenterates and Echinoderms. Coelenterate and echinoderm toxins range from small molecular weight amines, to sterols, to large complex carbohydrate chains, to proteins of over 100,000 daltons. Molecular size sometimes reflects taxonomy, e.g., sea anemones (Actiniaria) all possess toxic polypeptides varying in size from 3,000 to 10,000 daltons while jellyfish contain toxic proteins (ca. 100,000 daltons). Carotenoids have been isolated from Asterias species (starfish), Echinoidea (sea urchins), and Anthozoans such as Actiniaria (sea anemones) and the corals. These are sometimes complexed with sterols (J5). 

Two characteristics of the lyase that we have purified may be significant. First, the small molecular size of the protein may confer it a high mobility that could be helpful to its movement through host cell walls. In second place, it is an endo-type enzyme, fact that has been considered essential for maceration of plant tissues [35]. In this sense it is noteworthy that between the battery of pectic enzymes produced by FORL, this pectin lyase is the only protein that behaves as an endo-type. 

An important technique for the qualitative and quantitative analysis of different macromolecular materiafs is based on the efectrophoretic separation of particfes having different transport vefocities (e.g., because they have different zeta potentiafs). This technique is used for the anafysis of proteins, pofysaccharides, and other naturally occurring substances whose molecular size approaches that of colloidal particles (for more details, see Section 30.3.4). It is an advantage of the electrophoretic method that mild experimental conditions can be used—dilute solutions with pH values around 7, room temperature, and so on—which are not destructive to the biological macromolecules. 

Buchwald, P., Bodor, N. Octanol-water partition of nonzwitterionic peptides predictive power of a molecular size-based model. Proteins 1998, 30, 86-99. 

On-line dialysis also separates the analyte from tissue matrix based upon molecular size, but in this case, the sample extract is passed over a membrane filter through which the analyte (and other low molecular weight compounds) is diffused into a second solvent on the other side of the membrane filter. Usually, the second solvent is then concentrated on to an SPE column to minimize the dilution effect that is caused by the dialysis process. Agasoester used on-line dialysis to separate oxytetracycline from muscle, liver, milk, and egg tissue matrix components. A problem encountered with on-line dialysis is the inability of analyte molecules that are bound to proteins in the sample extract to pass through the membrane filter. Problems with membrane clogging are reduced with on-line dialysis compared with ultrafiltration because no external force is being applied to bring the analyte across the membrane filter. 

Melrose, J. and Ghosh, P, Determination of the average molecular size of glycosaminoglycans by fast protein liquid chromatography, ]. Chromatogr., 637, 91, 1993. 

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