[mrydberg]

I am performing enzymatic hydrolysis of glucuronated drug molecules in urine using beta-glucurodinase. The enzyme I have requires the pH to be buffered around 6.8, and I am using ammonium acetate for this. Right now I mix 50ul of 0.5M buffer solution with 50ul of urine and 10ul of my enzyme solution. I would ideally like to dilute my urine sample as little as possible, so how could I minimize the amount of buffer solution I need?

[Babcock_Hall]

That is a difficult question without knowing more about this enzyme, especially in regard to its preference regarding ionic strength.  What considerations made you choose ammonium acetate?  What about using one of the Good's buffers, such as MOPS or PIPES, both of which buffer effectively in this region?  BisTris is another go-to buffer for me in this region.

I would consider using a lower final concentration of buffer if possible.  That way you could work from a 5X or 10x concentrated stock and have less dilution.  One disadvantage of working from a concentrated stock is that the final pH will not be identical to the pH of the stock buffer (but some buffers have known correction factors).

[mrydberg]

Thank you for the response. I selected ammonium acetate because I had seen it in literature used for my enzyme (Roche E. coli). I had a look at Good's buffers and they look like they would work very well for me. They also have correction factors listed for some, so I could try to work backwards from where I need to be to where I should start. If I started with a buffer solution that was 1M instead of 0.5M, then I would add 20ul instead of 50ul to the urine sample if I accounted for the pH shift? It seems too simple, am I missing something?

[Babcock_Hall]

I am hesitant to tell people to do something other than what is in the literature, unless there is a good reason.  Obviously if there were other literature on this enzyme in different buffers, that would be helpful.  Borek would know more, but I don't that that ammonium acetate has much buffer capacity at this pH.  Other buffers that had more buffering capacity might be used at lower concentration.  If the enzyme prefers relatively high ionic strength, one might add KCl, to compensate for the hypothetically lower ionic strength of different buffer.

Regarding the pH shift, my previous comment was strictly about pH changes during dilution, not about any changes in pH due to the sample itself.  If I am following your proposal below, 25 µL of a 1 M stock is the same as 50 µL of a 0.5 M stock.

[mrydberg]

I really appreciate your help Babcock. What you say about using a more concentrated stock solution, and using a lower concentration of a buffer with higher buffer capacity makes sense. What is giving me pause is that in almost every methodology section I read in papers discussing enzymatic hydrolysis with beta-glucuronidase, they describe adding at least an equal volume of the buffer solution to the urine sample. Sometimes it can be a little less but it is never say five times less. I feel like I am missing something important about the process.

Hypothetically, if I buy a Good's buffer and make a concentrated solution, would this allow me to significantly reduce the volume of buffer solution I need to add to my sample (assuming it is compatible with the enzyme)?. I am ready to buy any enzyme and any buffer that would allow me to accomplish this goal, but I currently lack sufficient understanding to make decision.