Proteins molar mass determination

Molar mass determinations based on SDS-PAGE is sometimes misleading, since some proteins are not conversed completely into a rod-like shape or the protein/SDS ratio differs from the average. 

For characterization of a GPC medium other than defined macromolecules as dextrans, polypeptides, polystyrene sulfates, or colloidal gold may be used. For these standards the eluent is ddH20. These macromolecules are suitable for molar mass determination of proteins with restrictions only. 

Mass spectrometry has assumed great importance in determinations of the molar masses of biological macromolecules, even quite large ones. This is due to developments such as electrospray ionisation (ESI) and matrix assisted laser desorption/ ionisation (MALDI), which have made it possible to determine the molar masses of biopolymers up to several 100 kDa (Pitt 1996 Kellner et al. 1999 Snyder 2000). The combination of MALDI techniques with time-of-flight mass spectrometers (MALDI-TOF) is of particular significance for determination of the molar masses of proteins with high sensitivity (typically pmol quantities, although exceptionally fmol) and precision (proteins up to 100 kDa with precision of about 0.01 %). Mass spectrometry can provide very accurate measurements of protein molar mass that can yield information about even minor structural modifications not readily accessible by other means. 

On the other hand, denaturation by such reagents results in loosening of the original protein structure and the polypeptide chain configuration becomes a random coil which leads to changes in retention volume. This fact has to be taken into account especially in molar mass determinations. SDS is bound to protein propor-... 

A biochemist isolates a new protein and determines its molar mass by osmotic pressure measurements. A 50.0-mL solution is prepared by dissolving 225 mg of the protein in water. The solution has an osmotic pressure of 4.18 mm Hg at 25°C. What is the molar mass of the new protein ... 

C12-0018. When 7.50 mg of a protein is dissolved in water to give 10.00 mL of solution, the osmotic pressure is found to be 1.66 torr at 21°C. Determine the molar mass of the protein. 

In addition to the determination of molar mass distributions and various molar mass averages there are many experiments, requiring sometimes sophisticated data evaluation, that can be carried out with an analytical ultracentrifuge. Examples are the analysis of association, the analysis of heterogeneity, the observation of chemical reactions, and protein characterization, to mention only a few. A detailed discussion is beyond the scope of this article, but there is excellent literature available [77-79,81,87-89]... 

In this equation, u is the osmotic pressure in atmospheres, n is the number of moles of solute, R is the ideal gas constant (0.0821 Latm/K mol), T is the Kelvin temperature, V is the volume of the solution and i is the van t Hoff factor. If one knows the moles of solute and the volume in liters, n/V may be replaced by the molarity, M. It is possible to calculate the molar mass of a solute from osmotic pressure measurements. This is especially useful in the determination of the molar mass of large molecules such as proteins. 

A solution prepared by dissolving 6.95 x 10 1 3 g of protein in 0.0300 L of water has an osmotic pressure of 0.195 torr at 25°C. Assuming the protein is a nonelectrolyte, determine the molar mass of the gene fragment. 

In this form, van t Hoff s law of osmotic pressure is also used to determine the molar masses of biological and synthetic macromolecules. When the osmotic pressure is measured for a solution of macromolecules that contains more than one species of macromolecule (for example, a synthetic pol5mer with a distribution of molar masses or a protein molecule that undergoes association or dissociation), the osmotic pressures of the various solute species II, are additive. That is, in sufficiently dilute solution... 

Calibrate a GPC column for molar weight determination analogously. Use a set of defined proteins or polypeptides as standards (see inset in Fig. 3.4). If the mass differences allow a complete separation, the proteins can be applied as a mixture. 

It has been shown that the molar mass distributions of lignin sulfonates and kraft lignin can be determined by gel permeation chromatography. Calibration of the columns with lignin sulfonates of known molar mass or, alternatively, with commercially available proteins and polypeptides has been shown to give the same result. 

In biochemistry gel electrophoresis is the method of choice for the separation of various kinds of macromolecules (e.g. nucleic acids, proteins). It is also used in dendrimer chemistry for separation and as a method of determining relative molar masses and for qualitative assessment of the purity of a dendrimer sample. 

Osmotic pressure measurements are particularly convenient for determination of the molar mass of macromolecules such as proteins. 

One of the earliest methods for determining the molar mass of proteins was based on chemical analysis. A hemoglobin preparation produced from red blood cells separated from the blood by the use of a centrifuge was found to contain 0.335% iron, (a) If the hemoglobin molecule contains 1 atom of iron, what is its molar mass (b) If it contains 4 atoms of iron, what is its molar mass ... 

A solution of crab hemocyanin, a pigmented protein extracted from crabs, was prepared by dissolving 0.750 g in 125 mL of an aqueous medium. An osmotic pressure rise of 2.6 mm of the solution was detected at 4° C. The solution had a density of 1.00 g/mL. Determine the molar mass of the protein. 

Malate dehydrogenase from H. marismortui (AMDH) is the halophilic protein that has been studied most by solution structure methods. A molar mass of 87 kg/mol was determined for the native enzyme. It is stable at high concentrations of NaCl or KC1 and unfolds and dissociates below 2.5 M salt. Pundak and Eisenberg (1981) first measured values for the solvent interactions of AMDH and found that, in contrast to nonhalophilic globular proteins in similar conditions (Bi 0.2—0.3 g/g,B3 0.01 g/g),the halophilic protein bound... 

Desalting or buffer exchanges are often required between purification steps. At the laboratory scale, the protein solution is placed in a tube of a semipermeable polymer membrane immersed in the desired buffer. The membrane pore size determines the minimum molar mass of the compounds that are retained. Small molecules with a molar mass below the membrane cut-off will flow freely across the membrane until the osmotic pressure equilibrium is reached. Complete buffer exchange requires several changes of the dialysis liquid. The process should be carried out at a temperature around 4°C, to avoid loss of activity. 

Using the value of a determined above, the results of the standard assay made initially to check the enzyme activity, the assay in part C, and the given concentration of the enzyme stock solution in g L , calculate the specific activity of the enzyme— that is, the number of micromoles of sucrose hydrolyzed per minute per gram of enzyme present. (The specific activity of an enzyme preparation is of course a function of the purity of the enzyme. As inactive protein is removed from the preparation, the specific activity will rise. When the specific activity can no longer be increased by any purification method, a homogeneous enzyme preparation may have been achieved but proof of this depends on other criteria.) The exact chemical composition of invertase is still unknown, but its molar mass has been estimated at 100,000 g mol Combining this datum with your calculated specific activity, estimate the turnover number for the enzyme. 

Figure 4-7. Determination of the molar mass of globular proteins by gel filtration. There is a linear dependence between the elution volume (Ve) (usually expressed as the normalised ratio V e/V o, where V0 is the void volume) and the...

Figure 4-2S. SDS-PAGE determination of molar masses of proteins under denaturing conditions. The pattern of bands from a standard protein mix after SDS-PAGE is shown...

Gel filtration is frequently used to determine the molar masses of native proteins (Ch. 4.1.2.2). To obtain accurate results, it is important that there should be no nonspecific interactions between the protein and the gel matrix, and this can usually be assured by the right choice of buffers. The method is not very accurate, and it needs calibration with protein standards of known molar masses and of similar shape. If the protein has a characteristic activity that can be measured, e.g., an enzyme activity, then its molar mass can be determined using crude protein mixtures by assaying the eluate from a gel filtration column for activity. 

To determine the molar mass of a certain protein, 1.00 X 10 g of the protein was dissolved in enough water to make 1.00 mL of solution. The osmotic pressure of this solution was found to be 1.12 torr at 25.0°C. Calculate the molar mass of the protein. 

Another separation technique of particular application for proteins, high-molar-mass molecules, and particles is the general class known as field-flow fractionation (FFF) in its various forms (cross-flow, sedimentation, thermal, and electrical). Once again, MALS detection permits mass and size determinations in an absolute sense without calibration. For homogeneous particles of relatively simple structure, a concentration detector is not required to calculate size and differential size and mass fraction distributions. Capillary hydrodynamic fractionation (CHDF) is another particle separation technique that may be used successfully with MALS detection. 

Fundamental properties of macromolecules, such as viscoelasticity and flow behavior, primarily depend on the dimensions and the conformations of macromolecules. Primary biological functions substantially depend on the dimensions of natural macromolecules such as proteins and enzymes. Hence, a primary method to understand the physical properties of macromolecules, synthetic and natural, involves determination of the dimension as a function of the molar mass. A convenient method to fractionate macromolecules is the size-exclusion chromatography (SEC) technique. SEC fractionation is rapid and efficient and requires small amounts of the polymeric sample. A method to measure the dimension of the macromolecules as a function of the molar mass is an on-line technique to a SEC chromatographic system. 

Osmosis The tendency of a solvent to flow through a membrane into a more concentrated solution. It is used to determine an unknown molar mass, particularly for large molecules such as polymers and proteins. 

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