Amino acids isoelectric point, determination

The glycoprotein ceruloplasmin is a monomeric enzyme with a molecular mass of about 130 kDa and 1000 amino-acid residues. The carbohydrate content has been determined to be 10% and an acidic isoelectric point of 4.4 for human ceruloplasmin has been reported. 

Most of the E. coli ribosomal proteins are rather basic with high isoelectric points (Kaltschmidt, 1971) and a high content of basic amino acids (Tables I and II). The complete primary structures of all . coli ribosomal proteins have been determined by Wittmann-Liebold and coworkers (see Table III and Appendix). 

The molecular mass of the native AMDase was estimated to be about 22 kDa by gel filtration on HPLC. Determination of the molecular mass of denatured protein by SDS-PAGE gave a value of 24 kDa. These results indicate that the purified enzyme is a monomeric protein. The enzyme had an isoelectric point of 4.7. The amino acid sequence of the NH2-terminus of the enzyme was determined to be Metl-Gln-Gln-Ala-Ser5-Thr-Pro-Thr-Ile-Glyl0-Met-Ile-Val-Pro-Prol5-Ala-Ala-Gly-Leu-Val20-Pro-Ala-Asp-Gly-Ala25. 

The cell-bound amylopullulanase was solubilized with detergent and lipase. It was then purified to homogeneity by treatment with streptomycin sulfate and ammonium sulfate, and by DEAE-Sephacel, octyl-Sepharose and puUulan-Sepharose column chromatography (12). The final enzyme solution was purified 3511-fold over the crude enzyme extract with an overall recovery of 42% and had a specific activity of 481 units/mg protein. The average molecular weight of the enzyme was 136,500 determined by gel filtration on Sephacryl S-200 and SDS-PAGE, and it had an isoelectric point at pH 5.9. It was rich in acidic and hydrophobic amino acids. The purified enzyme was quite thermostable in the absence of substrate even up to 90°C with essentially no loss of activity in 30 min. However, the enzyme lost about 40% of its original activity at 95 C tested for 30 min. The optimum tenq)erature for the action of the purified enzyme on pullulan was 90°C. However, the enzyme activity rapidly decreased on incubation at 95°C to only 38% of the maximal 30 min. The enzyme was stable at pH 3.0-5.0 and was optimally active at pH 5.5. It produced only maltotriose and no panose or isopanose from pullulan. 

When they are first isolated, proteins are usually characterized by Mr, isoelectric point, and other easily measured properties. Among these is the amino acid composition112 which can be determined by completely hydrolyzing the protein to the free amino acids. Later, it is important to establish the primary structure or amino acid sequence.51 This has been accomplished traditionally by cutting the peptide chain into smaller pieces that can be characterized easily. However, most protein sequences are now deduced initially from the corresponding DNA sequences, but further chemical characterization is often needed. 

You will obtain a titration curve of an amino acid with a neutral side chain such as glycine, alanine, phenylalanine, leucine, or valine. If pH meters are available, you read the pH directly from the instrument after each addition of the base. If a pH meter is not available, you can obtain the pH with the aid of indicator papers. From the titration curve obtained, you can determine the pK values and the isoelectric point. 

Draw your titration curve. From the graph, determine your pK values and the isoelectric point of your amino acid. Record these on your Report Sheet. 

The following polar amino acid compositions with their internal pK values have been determined for two small proteins, lysozyme and calmodulin. Estimate their isoelectric points. 

Amino acids (and peptides) are characterized not only by their acid dissociation constants (pff,s), but also by an isoelectric point (pi), which is the pH at which the amino acid or peptide has no net charge. For a simple amino acid with only an a-carboxyl and an a-amino group the pi is determined by a balance between the tendency of the proto-nated amino group to lose a proton and the un-protonated carboxyl group to bind a proton. It can be readily shown that this pH is... 

Since the amide backbone of a protein is neutral and uncharged, the isoelectric point of a protein or peptide is determined by the relative numbers of acidic and basic amino acid residues present in the peptide. 

It must be pointed out that in general it is not possible to obtain an isoelectric point for a pure ampholyte, since equation (13) for the hydrogen ion concentration in the latter will be equal to that given by (14) for the isoelectric point in special cases only. It is usual, therefore, to add a small amount of alkali or acid in order to obtain an isoelectric solution. At the isoelectric point an amino-acid dissociates equally to yield RHt and R " ions, but if the hydrogen ion concentration exceeds the theoretical value for this point, there will be a tendency for the hydrogen ions to react with R- or RH= = to yield RH= = or RHj, respectively. In a solution of smaller hydrogen ion concentration, however, the RHt or RH" = ions will tend to ionize further. These conclusions have been utilized to determine the isoelectric points of amino-acids a small amount of ampholyte is added to each of a series of solutions of known pH these solutions should not be too strongly buffered. If there... 

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