Hi everybody,
I am planning to test effect of pH on endolysin (phage enzyme) activity, so I have to prepare solutions with different pHs. To delete effect of buffer components on enzymatic activity I have to one use universal buffer.
Can anyone help??
Thanks and appreciation in advance
You don't necessarily have to use a single buffer over the whole pH range. You can use a set of buffers with overlapping pH ranges. This allows you to test both the pH and the type of buffer, both of which can affect the enzyme activity.
Bis-Tris Propane can be used over a wide pH range because of its 2 different pKa, from about 5 to 10.5. It may not be the best buffer for the activity of your enzyme, however.
Each buffer system has its best buffering capacity at certain pH range; a deviation from this leads to lesser potency of the buffer to exert it's effect. So I think using different buffer systems, if well prepared will give a relatively accurate measure of the effect of pH on the enzyme activity with very little or no adverse influence from individual buffer components.
Hi there,
As Buffering is effective within 2 pH units centered on pKa, you will certainly have to use different buffer systems (ie. exploring a range of pH wider than 2 units). The main thing will be to have overlapping data between buffer systems in order to have a continous picture of the phenomenon over the whole pH range tested.
I've previously used a citrate-phosphate system to cover a wide pH range,
https://en.wikipedia.org/wiki/McIlvaine_buffer states that it should cover pH 2.2 to 8.
Hi again,
About the phosphate-citrate buffer recipe: be careful as both concentrations of phosphate and citrate vary with pH and therefore ionic strength does vary with pH too which may itself affect enzyme activity.
I am deeply thankful for your fruitful contribution. I will try different buffer systems with overlapping pH points.
You can not use a single buffer since the buffering capacity of each one best works within pKa +_ 1 . I advice you to read about the buffer & how you can choose the best one in Method of Enzymology volume 184. Good luck
What about using Britton–Robinson buffer
Britton–Robinson buffer (aka BRB aka PEM) is a "universal" pH buffer used for the range pH 2 to pH 12. Universal buffers consist of mixtures of acids of diminishing strength (increasing pKa) so that the change in pH is approximately proportional to the amount of alkali added. It consists of a mixture of 0.04 M H3BO3, 0.04 M H3PO4 and 0.04 M CH3COOH that has been titrated to the desired pH with 0.2 M NaOH. Britton and Robinson also proposed a second formulation that gave an essentially linear pH response to added alkali from pH 2.5 to pH 9.2 (and buffers to pH 12). This mixture consists of 0.0286 M citric acid, 0.0286 M KH2PO4, 0.0286 M H3BO3, 0.0286 M veronal and 0.0286 M HCl titrated with 0.2 M NaOH.
Thanks Dr. Yasser, I have already used this universal buffer system.
I had a similar problem where I studying the activity of an enzyme situated on a viral capsid, and I need to test pH ranges from 4-8 since it was thought this enzyme was active while entering through the endosomal pathway. Since we were using very sensitive biochemical and biophysical techniques, we needed a consistent buffer for each experiment to control for a possible allosteric changes that result from the buffer acting as a ligand. Thus , I went down the same rabbit hole as you looking for the ideal universal buffer, but I wasn't satisfied with any of the "known" universal buffer formulations. So I started testing my own formulations, which we published here (shameless plug)Article Universal buffers for use in biochemistry and biophysical experiments
.It was surprisingly difficult since every buffer brings its own caveat. For example, phosphate and citrate buffers are fantastic for temperature based experiments since pH doesn't change with temperature, while the pH of Tris based buffers decreases ~0.03 units/C. If your enzyme requires a divalent cation, phosphate and citrate buffers are not ideal.
To spare you reading the article, I attached a titration curve from the formulation I decided to use for all my experiments, which was 20mM HEPES, 20mM MES, 20mM Sodium Acetate (plus 120mM NaCl). Besides following Good's Criteria (https://en.wikipedia.org/wiki/Good%27s_buffers, Article Hydrogen Ion Buffers for Biological Research *
), it had a completely linear titration from pH2-9. This was super wild, because I could make 10x stock at pH4.00 and pH8.00 and mix them at a 1:1 ratio and get pH6.00 consistently and accurately. I used to have an equation I was used to determine the ratio for any specific pH I needed between pH4 and pH8, which worked so well I didn't even have to recheck it with the pH meter. I have long forgot the equation, but it wasn't terribly complicated and I can "re-derive" it if someone's interested.If you decide to use this buffer, this is what I did. For my 10X stock, I would mix 0.2M of Hepes, 0.2M of MES, and 0.2M of sodium acetate in 1L ddH2O (~0.6Mtotal). I would check the pH (it was somewhere around pH6.2 if I remember correctly), then split it into two separate bottles. One bottle would be used to titrate to pH8.00 and the other to pH4.00 making sure the molar amounts of HCl and NaOH added were similar between the two since you're adding salt. Then I would sit and mix combinations of those two in front of a pH meter until I got the pH I wanted ( I stopped doing this once I figured out the equation was super accurate). For my experiments, I would also add NaCl to have a final concentration of 120mM.
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