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Species |
Extraction Buffer |
Ammonium sulphate precipitation (%) |
Mangifera indica |
Tris-HCl 0.5M pH 6.4 |
80 |
Mangifera odorata |
Tris-HCl 0.2M pH 7.8 |
85 |
Durio brunei |
Phosphate buffer saline (PBS) 0.05 pH 6.8 |
80 |
Durio zibethinus |
Phosphate buffer saline (PBS) 0.02 pH 7.4 |
80 |
Syzygium aqueum (red flower) |
Tris-HCl 0.5M pH 6.4 |
80 |
Syzygium aqueum (white flower) |
Phosphate buffer saline (PBS) 0.02 pH 6.8 |
85 |
|
|
|
DISCUSSION
The first step to isolate this protein is to dissociate and solubilize the protein as the polypeptide chains in inclusion body protein are trapped in a number of partially or incompletely folded conformations that are stabilized by association with other polypeptide chain [15]. The selection of an appropriate buffer is important in order to maintain a protein at desired pH and to ensure reproducible experimental result. An appropriate buffer solution also can help improve the stability of protein molecules as these molecules are subjected to various forces designed to isolate them for study. A buffer solution can protect the integrity of the proteins while separating them from other integrated cell components [4, 9 and16]. The ammonium precipitation step was performed to concentrate the protein from dilute solution. This step is important since most of allergenic protein is hydrophobic and the saturation must be up to 80%. When high concentration of salt is present, protein tend to aggregate and precipitate out of solution. Since different proteins (especially for different species) precipitate at different salt concentration, salting out is often used during protein purification and this is actually the initial step of protein purification. Most protein will precipitate at 55% ammonium sulphate, thus seven concentrations with interval 10% and 5% saturation percentage up to 90% has been chosen to maximize the protein precipitation.
CONCLUSION
Choosing the right buffer is important to ensure reliable result of protein extraction. This study has shown that different buffer concentration and pH is critical in determining highest protein concentration yield for pollen protein from different genus. This study has also able to make recommendation for type of extraction buffer and percentage of Ammonium sulphate precipitation for each genus Mangifera, Durio and Syzygium. Different types of buffer definitely have different ability on the protein solubilization, while the 80% of salt precipitation was the most ideal percentage for salting out the protein in every species.
AcknowledgementThis research was funded Fundamental Research Grant Scheme (FRGS). The authors fully acknowledged Ministry of Higher Education (MOHE) and Universiti Malaysia Kelantan for the approved fund which makes this important research viable and effective. Authors acknowledge the immense help received from the scholars whose articles are cited and included in references of this manuscript. The authors are also grateful to authors /editors/publishers of all those articles, journals and books from where the literature for this article has been reviewed and discussed.
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