Hexokinase is an enzyme that phosphorylates hexoses, sugars known to have six carbons, creating hexose phosphate. In most animals, glucose is the highest important substrate for hexokinases. Its product is glucose-6-phosphate which is the most vital one. Hexokinase can move an inorganic phosphate group from ATP to a substrate.
Protein purification is always a very important feature in science in general, specifically biology. In this case, hexokinase purification will be looked at, and the process will be broken down to a point where it is stabilized or purified. I want to look at the purification of glucokinase which is among the four types of hexokinase. I would think of purifying glucokinase because it would be easy to identify.
Apart from identifying employing purification, it can also be easily identified due to the phosphate group. Basically, the enzyme glucokinase is found majorly in the liver of animals and in beta cells found in the pancreas. Beta cells yield and discharge the hormone insulin. The insulin hormone assists in regulating the rates of blood sugar in the body by regulating how much sugar is moved between the bloodstream and the cells as a source of energy to be used by the body.
The topmost concentration level of the enzyme glucokinase is mostly found in the liver of a living organism. This is because glucokinase is always wanted for glycogen synthesis, which always takes place in the liver where conditions favor this process having the highest concentration levels.
The first of all step of purification of an enzyme is to lay the foundations for acquiring the source. I will try and find out where glucokinase can be sourced from within the human body. Within this vital process, I need to obtain a sample known as Bacillus stearothermophilus, a microbial microorganism line that is most likely the suitable candidate because it is an extremely stable organism that favors the persistent extraction of glucokinase.
The first step of sourcing for glucokinase enzyme via this sample (Bacillus stearothermophilus) includes beginning by first tending to the enzyme. As pointed out in the PowerPoint, which acts as my guidelines in this process, culturing the enzyme is done at very extreme temperatures, approximately -500C.
The process of sourcing for the protein, which in this case is glucokinase enzyme, continues by involving the use of ammonium sulfate ((NH4)2SO4), which is involved in the process of fractionation. Gel filtration, also commonly acknowledged as size elimination chromatography, should be applied to distinguish proteins based on their molecular mass. A porous resin is an element usually used to carry out this separation process.
Thereafter conducting chromatography, the enzyme is in needs to be eluted by applying the use of sodium chloride (NaCl) as a gradient. Some fractions of kinase will still be available, and they will need to be removed further through ion exchange chromatography. There are various options to use: DEAE- Sephadex, SP-Sepharose, Bio-Rex70, and P cellulose. Instead, I will use DEAE- Sephadex in this process, a continuous cycle amounting to three times to cleanse the sample fully.
I am almost finalizing acquiring a pure sample of the enzyme glucokinase. During the final stages now, I do a process known as elution, which involves washing the column using sodium chloride (NaCl) to improve the ionic stability existing within the sample.
In the whole time amidst these steps, testing how pure the samples are is necessary, and it needs to be done regularly from time to time. The latest tendency seeks to encourage mass spectrometry, a parameter that seeks to measure the weight-to-charge proportion of ions to allow for the identification of the task the ions are involved in, which can be weighed up with the total protein to try and identify the distinct activity that is occurring. At the end of the process, I will mathematically compute the yielded percentage by scrutinizing the whole activity at a specific step and comparing it to the entire process right before the extraction.
After the whole process of purifying the glucokinase enzyme, I now get to the point where I have to prove if I have indeed extracted the said enzyme. In proving my extraction and purification, I can use glucose to transfigure the glucokinase from the protein specimen to glucose -6- phosphate. If the end product is recognized as glucose -6- phosphate, you can confirm that it is glucokinase, which is an undeniable fact that it is hexokinase.