The isoelectric point of a protein

The speed with which bovine serum albumin (BSA) moves through water under the influence of an electric field was monitored at several values of pH, and the data are listed below. What is the isoelectric point of the protein  

Strategy If we plot speed against pH, we can use interpolation to find the pH at which the speed is zero, which is the pH at which the molecule has zero net charge. 

Solution The data are plotted in Fig. 8.17. The velocity passes through zero at pH = 4.8 hence pH = 4.8 is the isoelectric point. 

In capillary electrophoresis, the sample is dispersed in a medium (such as methylcellulose) and held in a thin glass or plastic tube with diameters ranging from 20 to 100 pm. The small size of the apparatus makes it easy to dissipate heat when large electric fields are applied. Excellent separations may be achieved in minutes rather than hours. 

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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. 

To date, the most powerful combination of electrophoresis techniques employs IEF in the first dimension, resolving on the basis of pi, and SDS-PAGE in the second dimension to separate on the basis of molecular weight. The order is crucial, since the isoelectric point of a protein effectively disappears upon treatment with SDS, which yields a uniformly negative charge/mass ratio. 

The effect of the charged surface is closely related to the pH and ionic strength of the solution. For example, if the pH of the solution corresponds to the isoelectric point of a protein its net charge will be zero and as a result it will be transported unimpeded through a charged membrane. Decrease in ionic strength due to the loss of electrolytes from the solution during ultrafiltration results in an increase in interaction between the species themselves and with the membrane. Some of the above mentioned effects have been studied and discussed by Staub et al. (1984). 

The isoelectric point of a protein is defined as that pH at which the net charge is zero (Wismer-Pedersen, 1971). Since protein-protein ionic interactions are promoted at this point, it would be expected that the protein matrix would shrink and WHC would be at a minimum (Kapsalis, 1975). It follows that increasing the pH away from isoelectric point would also result in a higher WHC, since protein-water interactions are favored (Hamm, 1960). Bouton et al. (1971) were able to increase the ultimate pH and WHC of meat by preslaughter injection of epinephrine and showed that tenderness increased directly with pH values. Further work by Bouton et al. (1972) and Bouton and Harris (1972) showed that as pH increased from normal values of 5.5 to 7.0, tenderness of the tissue increased and became independent of the contracture state. 

However, the effect tends to be smaller in HIC than in ion exdiange. This is not unexpected. A dunge in pH will affect retention oidy if it affects the neighborhood of the hydrophobic area that binds to the suifece. Experiments did not conform with the expectation that retention should be greatest at the isoelectric point of a protein (10). 

Since the pH-gradients are remarkably linear, the isoelectric point of a protein can be determined fairly closely by means of markers. These consist of stained proteins with known isoelectric points. This possibility has been mentioned by Awdeh and co-workers (39), Wrigley (29), Leaback and Rutter (37,38) and others. 

A much better technique of protein purification is that of isoelectric focussing. Use is made of the isoelectric point of a protein. Suppose that the isoelectric point of a protein is 6.0. If we prepare a pH gradient in a column so that the pH varies from 1-10 and place all the proteins in the sample on top of the column and start the current, all the proteins will migrate dependent on the charge that they possess. At the space where pH of the gradient is 6, while other proteins will continue moving, the desired protein will become immobile since it possesses no net charge the pH is isoelectric for it. The method is discussed in reasonable details in Chapter 12. 

The isoelectric point of a protein varies with the environment. Consequently, whenever stating pi of a protein, it is a must to specify the buffer and the ionic strength of the solution in which the protein is suspended. 

In your notebook, make a drawing of the observed banding pattern, labeling both the hemoglobin and cytchrome C. Explain, in relevant detail, what you can determine about the isoelectric points of these proteins and what might happen in the gel if the buffer pH were changed from 7.9 to 3.9. 

Succinylation reduced the isoelectric point of yeast proteins from pH A.5 to 4.0 and markedly improved their solubility in pH range 4.5 to 6. Heat denatured yeast proteins were facilely solubilized following succinylation (74). Succinylated yeast proteins were very stable to heat above pH 5, and remained soluble at temperatures above 80°C. As the degree of succinylation was increased the rate of precipitation of the derivatized protein increased in the neighborhood of the isoelectric point and much larger protein floes were obtained facilitiating their recovery (74). 

Because the isoelectric points of alkaline proteins are generally in the range of pH 7 or above, coacervates whose composition includes alkaline proteins can be obtained at a more alkaline pH than when acid proteins are used. The greater the difference between the isoelectric points of the macromolecules, the more readily do they form coacervates. 

The generation of lactic acid through glycolysis produces a pH drop due to its accumulation in the muscle. The rate of drop may be faster or slower depending on the metabolic status of the muscle. In general, acid pH values (5.6-5.9) may be reached in just a few hours postmortem. Water binding decreases rapidly as pH approaches the isoelectric point of muscle proteins (pi values around 5.0). There is also a tightening of the structure and partial denaturation of myofibrillar proteins. 

Research into the isoelectric point of wine proteins has often been concurrent with smdies of wine protein size. Proteins with low isoelectric points (pi) were found to be significant contributors to total wine protein (Moretti and Berg 1965) and to wine haze (Bayly and Berg 1967). Hsu and Heatherbell (1987a) confirmed this observation and suggested that the majority of wine proteins had a pi of 4.1-5.8, whilst Lee (1986) suggested the major protein fractions of wine had a pi of 4.8-5.7. Dawes et al. (1994) fractionated wine proteins on the basis of their pi and found that the five different fractions all produced haze after heat treatment. Haze particle formation was found to differ between the fractions however, leading to a statement that other wine components, such as phenolic compounds, need to be considered to understand fully protein haze. 

Speakman and Hirst (1933) and Speakman and Stott (1934) consider that an isoelectric region rather than an isoelectric point should be recognized similar results are observed with silk (Howitt, 1946). Lemin and Vickerstaff (1946) claim to have determined the isoionic point of wool by measurement of the pH at which addition of salt does not affect the pH. They obtained a value of 6.2 which is approximately the midpoint of the isoelectric region. It is not possible to determine the isoionic point of a protein by measurements made, as in the present instance, at constant salt concentration. The true value could be obtained by carrjdng out the... 

If gel filtration procedures cannot be employed for removal of lactoperoxidase, the enzyme can be removed by taking advantage of the fact that it is a basic protein. It is easily separated from most proteins which have lower isoelectric points by the use of ion exchange resin. The reaction mixture is passed through a small column of IRC 50 at a pH above the isoelectric point of iodinated protein but below pH 8.0. This will remove the lactoperoxidase from the mixture without adsorbing proteins of lower isoelectric points. 

Rezwan, K., Meier, L.P, and Gauckler, L.J., A prediction method for the isoelectric point of binary protein mixtures of bovine serum albumin and lysozyme adsorbed on colloidal titania and alumina particles, Langmuir, 21, 3493, 2005. 

Since the isoelectric point of most proteins is between a pH of 4 and 5, it would be expected that operation in this pH range would result in the lowest gel concentration. Further, since changes in pH are not expected to change the... 

The isoelectric pH of a protein is the pH at which the protein is immobile in an electric field. At this pH, the protein exists as a zwitterion having equal number of positive and negative charges, the net charge being zero. The Isoionic point of a protein is defined as the pH at which the total number of H taken up by a protein is equal to the total number of H dissociated from it. Isolonic and Isoelectric point of a protein will essentially be the same if the protein does not bind ions other than H". Since this is an ideal condition and not usually obtainable, the Isoionlc and isoelectric points of a given protein differ. 

The titration curve of 3-lactoglobulin at various ionic strengths (Fig. 1.34) shows that the isoelectric point of this protein, at pH 5.18, is independent of the salts present. The titration curves are, however, steeper with increasing ionic strength, which indicates greater suppression of the electrostatic interaction between protein molecules. At its isoelectric point a protein is the least soluble and the most likely to precipitate ( isoelectric precipitation ) and is at its maximal crystallization capacity. The viscosity of solubilized proteins and the swelling power of insoluble proteins are at a minimum at the isoelectric point. 

The solubility of proteins near their isoelectric point environments can be improved by complexation with charged polysaccharide such as pectin, mainly due to electrostatic interactions. It was found that at pH values between 4 and 5, near the isoelectric point of soy protein, the protein particles have a lower net charge, causing aggregation, having no electrostatic repulsion forces, hydrophobic interactions are dominant [32]. 

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