Isoelectric point estimation

Thomson MOW Click Organic Interactive to learn to estimate isoelectric points for simple amino acids and peptides. 

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. 

For the SAXS studies a CBH II sample was prepared by affinity chromatography from r. reesei QM 9414 to give the enzyme in a homogeneous form 27. In SDS-PAGE the protein had a size of 58 kDa and the isoelectric point was 4.9. Glycosy-lation was estimated as 8 to 18 % 36. The molar absorptivity at 280 nm was 75 000 M xm To obtain the core protein partial proteolytic hydrolysis with papain was per-... 

The a-glucuronidase of T reesei also had an acidic isoelectric point (25). It had a molecular weight of about 70 kDa as estimated by gel chromatography and a pH-optimum at pH 6 with 4-O-methylgucurono-xylobiose as substrate. The very recently characterized a-glucuronidase of the thermophilic fungus Thermoascus aurantiacus (51) was a single polypeptide chain with a MW of... 

Hariita et al. (14) prepared spherical particles of molybdenum sulfide and cobalt sulfide with a narrow size distribution by reaction of dilute ammonium orthomolybdate or cobalt(II) acetate with sulfide ions liberated from thioacetamide as a reservoir of S2- ions in weakly acidic media. The compositions of these metal sulfides were estimated to be Mo S 0 = 1.0 1.7 3.0 and Co S 0 = 1,0 4.5 6.4 by chemical analysis. Figure 3.1.4 shows an SEM of a thus prepared uniform molybdenum sulfide particles sample. These sulfide particles were of no distinct crystal structure as shown by x-ray diffractometry. The isoelectric points of the Mo sulfide and Co sulfide particles in terms of pH were 1.9 and 3.1, respectively. Both of them are useful as hydrodesulfurization catalysts. 

Use these data to evaluate the isoelectric point for Th(OH)4. Since H+ and OH- appear to be potential determining, p may be estimated at various pH levels according to Equation (11.1) if we identify the isoelectric point with the true point of zero charge. Compare these values... 

The isoionic point of RNase-A determined as the pH of a concentrated salt-free solution is 9.60 (268). Estimates of the isoelectric point in buffers not containing phosphate all give values above 9, Anderson and Alberty (269) reporting 9.45. In phosphate buffers specific anion binding dramatically reduces the measured isoelectric point to an extent dependent on the total phosphate concentration. Values below 6 have been measured (270). 

Be able to solve acid-base problems involving amino acids, draw structures at various pH values, estimate isoelectric points and net charges, and construct and interpret their titration curves. 

Be familiar with the general structure of peptides and their synthesis and the properties of the peptide bond be familiar with methods of end-group analysis and be able to apply the concepts of acid-base chemistry to peptides, including estimation of net charges and isoelectric points. 

Figure 4.14 Estimation of the isoelectric point of human a-acid glycoprotein (orosomu-coid), an acid glycoprotein from plasma, pi is the point at which the mobility curve intersects the x axis. (Reproduced with permission from Bezkorovainy A. Basic Protein Chemistry. Springfield, IL Thomas, p. 22, 1970.)...

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. 

The native molecular mass of aphid myrosinase, estimated from gel filtration, is approximately 97 kDa, while the molecular mass of the denatured and reduced protein is 53 kDa, estimated from SDS PAGE, and the subunit by MALDI-TOF mass spectrometry 54 kDa 500 Da. Thus, aphid myrosinase appears to be a dimeric protein, with identical subunits. The isoelectric points (pi) of aphid myrosinase are 4.90 and 4.95, the latter isoform being the most abundant. 

Isoelectric focusing is a mature separation technique that has a place in any laboratory doing work with proteins. The analysis of a protein is not complete without a determination of its isoelectric point and all protein databases have at least estimates of the pis of the represented proteins. Proteins thought to be pure by other methods are often found to be mixtures of several proteins when analyzed by IEF. Isomeric forms of the same protein that are revealed by IEF are valuable indicators of mutations or differences in posttranslational modifications. IEF plays a crucial role in 2-D PAGE and preparative IEF allows for high-purity fractionations of unparalleled resolution. A simple keyword search of literature databases shows that about 500 journal articles are written per year referring to IEF. This amply attests to the value of IEF as a tool for protein analysis and purification. 

Fig. 3.25 presents the aqueous solutions in the absence of a surfactant at constant ionic strength (HC1 + KC1) [186,197], It can be seen that at pH > 5.5, op-potential becomes constant and equal to about 30 mV. At pH < 5.5 the potential sharply decreases and becomes zero at pH 4.5, i.e. an isoelectric state at the solution surface is reached. As it is known, the isoelectric point corresponds to a pH value at which the electrokinetic phenomena are not observed. Since in the absence of the potential of the diffuse electric layer, the electrokinetic potential (zeta-potential) should also be equal to zero, the isoelectric point can be used to determine pH value at which isoelectric state is controlled by the change in pH. This is very interesting, for it means that the charge at the surface of the aqueous solutions is mainly due to the adsorption of H+ and OH" ions. Estimation of the adsorption potential of these ions in the Stem layer (under the assumption that the amounts of both ions absorbed are equal) showed that the adsorption potential of OH" ions is higher. It follows that ( -potential at the solution/air interface appears as a result of adsorption of OH" ions. 

Immunoprints with a panel of 19 sera from SPT-reactive subjects to Calvatia cyathiformis crude extracts revealed at least 21 different protein-binding bands [48]. Two of these proteins (pi 9.3 and pi 6.6) reacted with 68 and 63%, respectively, of the sera tested. The most reactive allergen had an estimated isoelectric point (pi) of 9.3 and was particularly labile [49]. Preparative isoelectric focusing was used to isolate the pi 9.3 allergen, which has a molecular weight... 

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