Isoelectric Point Problems

[Bowie Maur]

NoAmino acids are acids. They are also bases containing an amino group. The term amphoteric is often used to describe amino acids, meaning that they are capable of acting as both acids and bases.

If the pH is decreased to a low enough value (e.g. pH 1) then the carboxylate salt will be protonated to give the neutral carboxylic acid, and the molecule will have a net charge of +1.

For a typical amino acid, there will be a range of pH values where the positively charged form dominates, another where the neutral form dominates, and finally one where the negatively charged one dominates.

If only there were some formula we could use for figuring out the pH of points A and B on the graph above, where the acid and its conjugate base are present in equal concentration.

There is more to isoelectric point than just the calculation of pI values for individual amino acids! The same concepts also apply to peptides and proteins, each of which will have a pI value that is influenced by the characteristics of its side chains.

We address the importance of the isoelectric point (IEP) of proteins and membrane components such as phospholipids for our understanding and interpretation of isoforms and opposite charge interactions in the formation of complexes. Five examples drawn from the literature are newly approached from the IEP point of view to clarify general principles.

The isoelectric point (IEP) of a protein is generally considered to be a mere physicochemical property that results from the summed ambivalent charge properties of its amino acid constituents. In fact, the IEP values of proteins are often not even recorded in data books, despite the primary structure of the protein being fully known. We show here that the IEP is a powerful tool to predict and understand interactions between proteins, proteins and membranes (phospholipids) or to determine the presence of protein isoforms.

The calculated isoelectric point (IEP) of the rainbow trout hormone stanniocalcin as a function of deamidation of N-residues. The primary structure of the mature rainbow trout stanniocalcin polypeptide is derived from the SwissProt data bank under accession no. P43648

The biochemical consequence of this decreased IEP is not yet clear, but it may be a signal for metabolic clearance of the hormone that should have a short half-life to fine-tune the Ca2+ influx under variable and varying conditions. To acquire further insight, one should know the IEP of the hormone receptor as it is conceivable that when the IEP of receptor and activator approach each other, their mutual attraction will be minimised. An example of this principle will be given in the third case study presented in a following section (repulsion of phosphorylated phospholemman by the near-isoelectric Na,K-ATPase) below. This concept should be taken into consideration in future evaluations of the function of the stanniocalcin receptor, which awaits molecular demonstration. Only circumstantial evidence is currently available that stanniocalcin signals through a G-protein-coupled plasma membrane receptor (Flik 1990).

Another pertinent example of charge interaction between functionally active proteins is the binding of α-MSH to the MC-R1, which occurs through the stepwise activation of adenylate cyclase, protein kinase A and tyrosinase to the final formation of the skin pigment melanin (Rozaud and Hearing 2005) and its rapid dispersion in the cell in background adaptation. A revealing recent finding is that the functional difference in teleost skin pigmentation (dorsal dark, ventral light) is due to a ventral localization of the melanocortin antagonist ASP (Agouti-Signaling Protein) (Cerd-Reverter et al. 2005). This has lead to the interesting question of whether the IEPs of agonist (α-MSH), antagonist (ASP) and receptor (MC-R1) have a predictive value for their interaction.

Step 1 would be the delivery of two electrons by the respiratory chain before a site of phosphorylation and step 2 would consist of the removal of two electrons immediately behind this site of phosphorylation. The final step of H+ transport from the intra-mitochondrial space to the outside compartment and vice versa may take place via E119 (isoform 1) or E126 (isoform 2) of the DCCD-binding protein. It should be noted that the involvement of disulfide bridges in energy-linked H+ transport has been suggested earlier by studies on triphenyltin inhibition of CF0-catalysed transmembrane H+ transfer in chloroplasts (Gould 1978). The construction of the proton gradient takes time, and this can be reduced by addition of the SH-reagent dithiothreitol (Schuurmans Stekhoven et al. 1970).

Although the interaction between FB and the DCCD binding protein is probably not of an electrostatic character, but rather of a reversibly covalent (disulfide bridging) one, the IEP considerations have also been decisive in coming to this conclusion in this example.

However, we cannot say that the subject of the IEP in relation to charge interactions is neglected in most publications. An example to the contrary is the elucidating explanation of the mechanism of interaction between cytochrome c reductase and cytochrome c oxidase with the intermediary cytochrome c (Stryer 1995). Another example is the release of phospholemman from the endoplasmic reticulum to the plasma membrane upon charge neutralization at the C-terminus by protein kinase C (PKC)-linked phosphorylation (Lansbery et al. 2006). Undoubtedly, other examples can also be found.

Open Access This is an open access article distributed under the terms of the Creative Commons Attribution Noncommercial License ( -nc/2.0 ), which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.

The isoelectric point (pI) is the pH at which a protein has no net charge. Ion exchange chromatography takes advantage of the fact that the relationship between net surface charge and pH is unique for a specific protein.

A protein that has no net charge at a pH equivalent to its isoelectric point (pI) will not interact with a charged resin. However, at a pH above its pl, a protein will bind to a positively charged ion exchange chromatography resin or anion exchanger. At a pH below its pI, a protein will bind to a negatively charged IEX resin or cation exchanger (Fig 1).

Weak ion exchangers can take up or lose protons with changing pH. As a result, their ion exchange capacity varies with pH. An advantage of a weak ion exchanger, such as DEAE (anionic), ANX (anionic), and CM (cationic) is that they can offer a different selectivity compared to strong ion exchangers.

The pI of the protein determines the buffer pH and the ion exchange column to use.At a buffer pH above its pI, a protein is negatively charged and will bind to an anion exchanger.At a buffer pH below its pI, a protein is positively charged and will bind to a cation exchanger.

The choice of buffering ion is critical to protein binding. When a buffer contains the wrong buffering ion, it can prevent binding of the target protein to the resin. Therefore, choose a buffering ion with same charge as the resin to prevent it from interacting with the ion exchange process.

An exception to this rule is with the use of phosphate buffers with anion exchange separations. However, phosphate buffers used in anion exchange must be prepared carefully to ensure reproducibility between runs.

Figure 3 lists commonly used buffers for anion- and cation-exchange chromatography. Our recommendation is to use a buffer concentration that is sufficient to maintain buffering capacity, constant pH, and with an ionic strength sufficiently low. A buffer concentration of 20 to 50 mM usually meets these needs.

Hi I print Liquid Light on steel plate all the time. I coat the plate with a single coat of poly urethane to create a surface for the emulsion to adhere. The technology here is about the urethane creating micro pours surface for the image to adhere to.

Regards acetic acid, this is a definite no, the emulsions that are in use here are sensitive to non buffered acid reactions. Instead just use a bath of your fixer as a stop bath then use the powdered fixer. By the way, I use the fixer at half the strength called for by the (Kodak) directions and I use three baths of fixer after my fixer that is acting as stop bath. You will go through a lot because it is diluted but that is perfectly all right. If you get that worry feeling about the fixer and the whole thing regarding image etch and stability then you can always turn to the classic PLANE THIOSULFATE FIXER, essentially this can be found in any good manual on salted paper printing. A silver image in a PLANE THIOSULFATE FIXER will not attack the formed silver image, it only affects the soluble silver complexes. The instructions offered with the Liquid Light product are correct in all regards it is just you will need 20 years experience in Non-standard silver processes to be able to understand what they are trying to say and what is implied between the lines.

I suggest that some of you might benefit from my Workshop in emulsion making and coating. In it, I will draw on my more than 30 years experience in Kodak R&D to explain some of the 'unknowns' you are experiencing as problems. I think it will also explain the reasons behind some of my answers. It starts on the 18th of June at the Formulary. See the other posts and links here for more information.

One simple answer, easy to give here, is that a stop bath is not unbuffered, but rather is a buffered solution and will gently lower the pH of the swollen gelatin to its proper level, (about 4.5) which is not only the isoelectric point of bone gelatin (photo grade) but also the point of minimum swell. This is the ideal position to be in for hardening to take place.

I was asking about the aluminum and which layer was lifting because of some aspects of commercial anodized aluminum. The anodized layer, particularly if it's been colored/dyed, is often treated with additional materials to enhance it's appearance and durability. These additional materials could prevent good adhesion of the emulsion (like trying to get the emulsion to stick to wax). A stronger or harsher detergent might be able to remove enough of the material. Sodium Hydroxide will strip the anodizing, so it should be avoided (read labels). A sulfuric acid wash (NOT hydrochloric) might remove the added materials, but may also destroy coloring dyes and can be nasty to work with.

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