Introduction
The isoelectric point, also referred to as the zwitterion's pH, signifies the pivotal moment when a molecule attains a neutral charge. Picture this: no positive or negative charges prevail, as an equal count of these charged entities coexist harmoniously.
When we focus specifically on amino acids, this moment occurs precisely when the molecule's positive and negative charges are perfectly balanced, a delicate equilibrium dictated by the pKa values assigned to the amino and carboxyl groups.
To fully grasp the intricacies of this concept, it is paramount to acknowledge that amino acids form the fundamental building blocks of proteins. These amino acids possess a dual nature, with an amino group (-NH2) on one end and a carboxyl group (-COOH) on the other.
Zwitterion
These groups exhibit characteristics of weak acids and bases, respectively, and their protonation or deprotonation hinges on the pH of their surroundings. At lower pH levels, the amino group happily embraces protonation (NH3+), while the carboxyl group resists such a transformation (COO-), resulting in an overall positive charge for the molecule. In contrast, at higher pH levels, the amino group stands proud as it sheds its proton (NH2), while the carboxyl group welcomes protonation (COOH), engendering a negative charge for the molecule. However, at the magical isoelectric point, the amino acid stands in its glory as a zwitterion, gracefully adorned with both positive and negative charges.
The isoelectric point serves a significant purpose, serving as a metric to gauge a molecule's acidity or basicity, elements that profoundly influence its solubility, stability, and activity.
This value also wields the power to prognosticate the behavior of proteins under diverse conditions, envisioning scenarios involving chromatography or electrophoresis with remarkable accuracy.
Amino Acid Isoelectric Points
The isoelectric point of an amino acid is influenced by its chemical structure, specifically the side chain (R group) that determines its properties. There are 20 common amino acids, each with a unique side chain, and their isoelectric points range from 5.5 to 12.0. The table below lists the isoelectric points of some of the most common amino acids:
Each amino acid has a different isoelectric point, which is influenced by the number and type of charged groups present in the molecule. Amino acids with acidic side chains, such as aspartic acid and glutamic acid, have low isoelectric points, while those with basic side chains, such as arginine and histidine, have high isoelectric points.
Meaning
At the isoelectric point, the molecule has no net charge, which means it is not attracted or repelled by charged particles in the environment. This may affect its solubility, as charged molecules tend to dissolve more readily in polar solvents.
If the pH of the solution is below the isoelectric point of the amino acid, the molecule will have a net positive charge and will be attracted to negatively charged surfaces, making it less soluble.
Conversely, if the pH is above the isoelectric point, the molecule will have a net negative charge and be repelled by a negatively charged surface, also decreasing its solubility.
In addition to solubility, the isoelectric point can affect the behavior of molecules during chromatography and electrophoresis. These techniques rely on the separation of molecules based on their charge and size, and the isoelectric point can be used to predict where molecules will move in these systems.
For example, during isoelectric focusing, proteins are separated based on their isoelectric point in a pH gradient. As proteins move through the gradient, they will reach a region where the pH is equal to their isoelectric point, causing them to stop moving because they have no net charge and thus can be separated based on the protein's isoelectric point.
How to Calculate Isoelectric Point
Step 1: Gather the Necessary Information
Before embarking on the calculation, ensure you have the essential data at hand. Specifically, you will need the pKa values of the amino group (-NH2) and the carboxyl group (-COOH) for the amino acid in question. These pKa values reflect the acidity constants associated with the protonation or deprotonation of these functional groups.
Step 2: Identify the Acidic and Basic pKa Values
From the gathered information, identify the pKa value that corresponds to the amino group's acidity (pKa1) and the pKa value associated with the carboxyl group's basicity (pKa2). The pKa1 typically represents the acidity constant for the amino group, while pKa2 indicates the basicity constant for the carboxyl group.
Step 3: Determine the Isoelectric Point
To calculate the isoelectric point, you need to find the pH at which the molecule possesses a net charge of zero. This means that the positive and negative charges within the molecule are perfectly balanced. The isoelectric point (pI) can be determined using the following formula:
pI = (pKa1 + pKa2) / 2
This formula takes the average of the pKa values associated with the amino and carboxyl groups.
Step 4: Interpret the Result
The calculated pI value represents the pH at which the molecule is electrically neutral. If the pH of the environment surrounding the molecule is below the pI, the molecule will carry a net positive charge. Conversely, if the pH is above the pI, the molecule will bear a net negative charge. Understanding the behavior of the molecule at different pH levels is crucial for predicting its solubility, stability, and other properties.
Step 5: Consider Other Factors
While the pKa values are fundamental in calculating the isoelectric point, it's important to note that there may be other factors that can influence the overall charge of the molecule, such as the presence of additional functional groups or modifications. These factors may require further considerations or adjustments in the calculation process.
Isoelectric Focusing Electrophoresis
Isoelectric focusing (IEF) is a molecular technique used in labs to separate different molecules based on the difference in their pI or isoelectric point. The technique is also known as electrofocusing.
Basically, it is a type of zone electrophoresis that uses the pH of the gel to determine the overall charge on the molecule of interest. In the first step, the proteins are separated by their pI value and then SDS-PAGE is used to separate them further based on their molecular weight.
In this technique, the separation of proteins is done by applying an electric field within the pH gradient. As the molecules move through the gradient in response to the voltage, they start to separate based on their size.
Upon reaching a pH value that matches its pI, a protein’s net charge becomes neutral, and it stops migrating. Thus, each protein in the sample is focused according to its pI.
IEF or Isoelectric focusing can be performed by using:
Carrier ampholytes that migrate through the gel to generate pH gradients.
Immobilized pH gradients (IPG) with ampholytes covalently bound to an acrylamide gel.
Isoelectric focusing (IEF) is widely used in molecular biology labs and biotech labs because it has better resolution and quantitation than the gel electrophoresis technique. Further, it’s easier to perform as there’s no stress of the placement of samples.
Today, many types of IEF have been developed, some of which include:
1.Microfluidic chip-based IEF: Electrophoresis using microchips offers a number of advantages over capillary electrophoresis, including:
Rapid protein analysis
Easy integration with other microfluidic units
Whole channel detection
Nitrocellulose films
Smaller sample sizes
Lower fabrication costs.
2.Capillary isoelectric focusing: A high-resolution method of separating molecules based on their pI point in fused-silica capillaries with internal diameters of 25-75 μM. The workflow can be easily automated in labs.
3.IEF-gel electrophoresis: A widely used technique in isoelectric focusing (IEF). It effectively minimizes convection and incorporates a gel-sieving effect to achieve precise separation of proteins according to their size.
In this blog, we will cover more about isoelectric focusing, including its working mechanism, its applications, and the industries that frequently use techniques.
IEF working
Isoelectric focusing is a technique that works by applying an electric field to the pH gradient medium containing protein samples.
Initially, proteins start moving towards their electrode with the opposite charge. However, as soon as they start migrating through the pH gradient gel matrix, they either lose or pick up protons, which results in a decrease in their electrophoretic mobility and net charge. Thus, the proteins start slowing down and eventually stop after arriving at the isoelectric point.
If proteins diffuse in a region with pH values lower than its pI, they become protonated and move toward the cathode in the presence of the electric field. Whereas, if proteins move to a region with greater pH values, they become negatively charged and move toward the anode. In this way, the separated proteins are focused in a sharp band in the pH gradient based on their respective pI values.
In the pH gradient medium, proteins move at different rates toward their pI values. However, once reached, they stick to those pH values for an extended time. Further, in IEF proteins reach their steady-state positions from anywhere in the gel.
To separate focused proteins based on their molecular weight, the gel is incubated in an SDS buffer and applied on SDS–PAGE slab gels. In addition to micro-range IPG strips (of different lengths and pH ranges), electrophoretic cells are used to achieve the highest resolution in IEF results.
The relative focusing power of two strips with the same length but different pH ranges can be determined by comparing the ratio of their pH ranges in terms of the number of pH units.
Usage
Isoelectric-focusing electrophoresis plays a crucial role in a variety of life sciences fields, spanning protein analysis, purification, proteomics research, and clinical diagnostics. It provides valuable insights into the separation and characterization of biomolecules based on their charge.
Molecule Separation
IEF is used to separate proteins in a mixture (mostly peptides, proteins, and different amino acid sequences) based on their pI values in a pH gradient gel matrix in the presence of an electric field. It’s a major procedure involved in the first dimension of a 2-D electrophoresis experiment. The second dimension of protein separation is accomplished by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
The preparative IEF method is used to isolate proteins from contaminants with slightly different pIs at the end of the purification process. It is usually used to isolate isoforms of proteins. Even small charge differences in the amino acid residues of proteins can be detected by using IEF.
IEF in analytical applications serves multiple purposes, including the evaluation of protein extract complexity and the identification of specific components. However, its exceptional resolving power makes it particularly well-suited for detecting microheterogeneity within purified proteins.
A combination of IEF on agarose gels and immunoblotting is now accepted as the gold standard for detecting oligoclonal Igs.
Biopharmaceutical
IEF is extensively used in the study and diagnosis of many genetically transmitted disorders by identifying abnormal proteins associated with the disorder.
Further, IEF is frequently employed by immunochemists to analyze a wide range of antigens and preparations. When combined with immunoblotting, this technique proves highly valuable in determining the specific antigen-binding profile of an antiserum or monoclonal antibody, enabling precise characterization.
Agriculture
IEF is widely used in soil science to identify matrices in organic fertilizers and assess organic matter stabilization in soil and composts. It provides valuable insights into soil health and nutrient management.
Further, IEF has profound applications in protein and enzyme analysis of oilseeds, cereals, plants, and vegetables. Its utilization in genetic studies has played a crucial role in enhancing the nutritional value of these seeds, driving advancements in agricultural and food science.
Advantages
Due to the pH gradient, high resolution is achieved.
Proteins that differ by 0.001 pH units in isoelectric pH can also be separated.
Disadvantages
Electrophoretic matrix has to be cooled several times.
Since the carrier ampholytes are used in a high concentration, a high voltage (up to 2000V) has to be applied.
Applications
For separation and identification of proteins and peptides.
For the determination of the isoelectric pH of proteins.
For research in enzymology, taxonomy, cytology, and immunology.
