For Proteins of Higher Molecular Weight

Materials Required Solution (1) Dissolve 10 mg of insulin in 1 ml of the mobile phase Solution (2) Dilute 100 i/ of solution (1) to 10 ml with the mobile phase and Solution (3) Dissolve 10 mg of procine insulin EPCRS of bovine insulin EPCRS, as appropriate, in 1 ml of the mobile phase. 

Inject 5 0 pi of each solution. Adjust the sensitivity of the detector so that the height of the principal peak in the chromatogram obtained with solution (2) is 50-70% of full-scale deflection. In the chromatogram obtained with solution (1) the sum of the area of any peak eluting before the principal peak is not greater than the area of the principal peak in the chromatogram obtained with solution (2) (1.0%). 

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Human Insulin For proteins of higher molecular weight... 

HUMAN INSULIN FOR PROTEINS OF HIGHER MOLECULAR WEIGHT... 

Size Exclusion Chromatography (Chapter 31) has also been included as a means of analysis for substances that undergo separation more or less as per their molecular size, viz., insulin and human insulin—for proteins of higher molecular weight corticotrophin—for impurities of higher molecular weights and plasma-protein solution—for polymers and aggregates. 

The enzymatic degradation of insulin was also shown to occur in the cytosol of alveolar cells, the pH optimum of the proteases being 7.4 [38]. To what extent intracellular proteases play a significant role in limiting the absorption of insulin is not clear, since the size of insulin likely allows paracellular transport over the alveolar epithelium. However, for proteins of higher molecular weight, that require transcellular transport, these proteases might certainly limit bioavailability. 

The elution volume, F/, and therefore the partition coefficient, is a function of the size of solute molecule, ie, hydrodynamic radius, and the porosity characteristics of the size-exclusion media. A protein of higher molecular weight is not necessarily larger than one of lower molecular weight. The hydrodynamic radii can be similar, as shown in Table 4 for ovalbumin and a-lactalbumin. The molecular weights of these proteins differ by 317% their radii differ by only 121% (53). 

In MALDI MS, the analyte is additionally embedded in higher molar excess of matrix molecules, which allows a soft desorption and ionization of the analyte. Typical molar ratios between the matrix and the analyte for measurement of low-molecular weight compounds (<500 Da) are in the range between 10 1 and 100 1, for higher masses (e.g., peptides and proteins) typical ratios are in the range of 1000 1 up to 100,000 1. Despite the capability to be measured by LDI MS, most classical ILs based on pyridinium... 

Plastocyanin consists of a single polypeptide chain of molecular weight around 10 500 and one copper atom. It is synthesized in the cytoplasm as a precursor of higher molecular weight,911 with an additional polypeptide that is essential for transport of the protein into the chloroplast. Within... 

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