Proteins are defined as the large molecule that is composed of one or more chains of amino acids in specific manner. Protein's study is very vast and divided into many areas such as food processing and manufacturing, biotechnology, biology and mostly important in the medicine. In the vast studies of proteins "Adsorption study of protein" is very popular and complicated. (1) Adsorption is a surface phenomenon means it is related with interaction of any surface like - solid - solid interaction, solid - liquid interaction and liquid - liquid interaction etc.
As we mentioned earlier, Adsorption of protein is very complicated study because here we going to study the adsorption of protein on to the solid surface in the water and protein shows folding and unfolding nature in the water. Adsorption phenomenon based on a principle that is whenever an interface formed (1)
between two different phases it has a high free energy than bulk phase. So the interface becomes thermodynamically stabilized by adsorbing other substance different than the solvent molecule. But in specific, the adsorption of protein on to the solid surface is depend on the structure and change in conformation of protein, hydrophobic and hydrophilic interactions, and the charges on their surface. (1)
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The proteins are large amphiphatic molecules this characteristic make them intrinsically surface active molecule and it causes a surface interaction. The main causes for the surface interaction are conformational entropies and restricted nobilities, intermolecular forces, Lewis acid - base forces, columbic forces, hydrophobic interactions, van der Waals forces.(3,4) additionally intramolecular forces within the protein molecules that might cause alteration of protein confirmation. The determination of extent of conformational changes is very difficult and even more its prediction. So for determination of protein adsorption kinetic study of the adsorption process is required. (5)
The above graph is plotted between initial concentration and adsorbed amount of BSA onto the surface of MCC at pH 7. The previously mentioned adsorption of BSA on MCC is also at pH 7 but the previous work done with the ratio of BSA conc. and MCC conc. it was 0.036 and there is no adsorption at all that's why in this case the ratio is increased to 1, 2 and 3. That means only BSA conc. has increased and MCC concentration remained same.
As the conc. of BSA increased there is well mannered adsorption seen. The graph shows as we increase the conc. of BSA the adsorbed amount also increases and will form a plateau. Higher conc. leads to higher adsorption because of electrostatic repulsive forces between two protein molecules. Due to loads and loads of BSA conc. each molecule repels each other because of same charge on surface. But then these molecules competitively bind on the surface of MCC. Actually the surface of MCC and the surface of BSA have same charges on their surface but for BSA, the surface of MCC is different and mainly adsorption takes place due to the hydrophobic interactions between both surfaces.
Graph 7: Initial concentration of BSA with Fractional coverage area of MCC at pH 7
The above graph plotted between initial concentration of BSA and fractional coverage of the MCC surfaces. The graph clearly shows as the concentration increase fractional coverage area also increases. But the time at which all adsorption sites are blocked or occupied by the BSA molecules the fractional coverage area remains constant and it forms a plateau means no increase in fractional coverage area after that. An option is there for increase in further fractional coverage area is Desorption.
The main objective of this work is to study the adsorption of protein on to solid surfaces at different conditions such as pH, media, ionic strength etc. the above discussed work is at pH 6 and 7. Adsorption of protein on to the surface varies at different pH values. As earlier said this work is carried out with other pH also that is 4 and 5. Basically the adsorption of protein governed by two forces hydrophobic and electrostatic interactions between protein and the solid surface.
The data obtained at pH 6 and 7 is far different than the pH 4 and 5. The pH and ionic strength affects adsorption on both MCC and silica surface. Another key of adsorption is "Iso-electric point" of protein (BSA) which is in the range of 4.7 - 5.1 so, BSA adsorption occurs in between this range. Hence, there is high adsorption of BSA on to MCC at pH 5. Also at pH 4 BSA showing a much higher adsorption than at pH 5. On the other hand at pH 6 and 7 there is no adsorption at all. This adsorption on to the MCC surface is governed by the electrostatic interaction.
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The further addition of 0.09% of sodium chloride also affects the adsorption of protein. It sometime supports the adsorption and sometime not. Because it depends on the distribution and interaction of salt ions that is, Na+ and Cl- with the both surface. So depending on the distribution of salt ions attraction and repulsion occurs.
On the other hand adsorption of BSA on silica surface occurs at all pH 4, 5 and 6. Here also hydrophobic and electrostatic interaction supports the adsorption. At pH 4 and 5 adsorption governs by the opposite charges of both surfaces. But at pH 6 the adsorption assisted by the hydrophobic interaction. The further addition of 0.09% of sodium chloride supports the adsorption of BSA on to silica surface at all pH.
Study of adsorption at higher concentrations of BSA also carried out. The study shows higher adsorption of BSA on MCC at all 4, 5 and 6 pH this adsorption particularly assisted by the hydrophobic interactions.
Conclusion . . . . . . . .
Overall the two factors hydrophobic and electrostatic interactions play an important role in the adsorption of BSA on to the surface of MCC and silica. These forces and the results obtained at different pH leads to following conclusion -
Adsorption of bovine serum albumin to microcrystalline cellulose was determined as function of protein concentration and pH of its aqueous solution. The maximum adsorption value attained at the BSA iso-electric point.
The overall adsorption was governed by hydrophobic and electrostatic interactions it sometimes supports the adsorption and sometimes not depends on the pH and ionic strength of the solution.
Adsorption of BSA on to the surface of silica is very well mannered and the adsorption attained at all 4, 5 and 6 pH. And the adsorption at 6 pH assisted by hydrophobic interaction instead electrostatic.
As the ratios of BSA and MCC increased up to 1, 2 and 3 the adsorption values were also increased and attained at all pH. Where, the adsorption has not seen at pH 6 at lower concentrations of BSA.