Question

I prepared a solution of n-benzoyl L-tyrosine p-nitroanilide (BTNA) in dimethyl sulfoxide for use in an enzymatic assay. However, mixing it with a solution of phosphate buffer produced a color change for unknown reasons. Here is what I did:

A 1.4mM solution of BTNA in DMSO was prepared.
5mM phosphate buffer was prepared. This was done by dissolving dipotassium phosphate in distilled water and adjusting the pH to 7.59 by addition of hydrochloric acid.
Into a cuvette was added 0.4mL of the BTNA solution, followed by addition of 0.1mL of the phosphate buffer solution.
When the BTNA solution and phosphate buffer were mixed, the mixture took on a yellow color. I tried mixing the BTNA solution with distilled water without any phosphate added to see if, perhaps, the act of mixing water with the BTNA solution is responsible for the color change. In this case, the mixture did not change color.

My question is thus: Why did mixing 0.4mL of BTNA solution in DMSO with 0.1mL of 7.59 phosphate buffer result in the mixture taking on a yellow color, whereas mixing BTNA solution with water did not result in a color change?

Sidenote: None of the solutions were tested for contaminants.

Answer 1

The yellow color observed when mixing BTNA solution in DMSO with phosphate buffer may be due to a chemical reaction, potentially involving hydrolysis of BTNA. Further experiments and analysis are needed to fully understand the cause of the color change.

Step-by-step explanation:

The color change that occurred when you mixed the BTNA solution in DMSO with the phosphate buffer is likely due to a chemical reaction between the two compounds. The yellow color suggests the formation of a new compound or a change in the structure of BTNA. One possibility is that the phosphate buffer caused a hydrolysis reaction in BTNA, leading to the formation of a yellow product.

In contrast, when you mixed the BTNA solution with water without any phosphate added, there was no color change. This suggests that the phosphate buffer is the cause of the color change and not simply the act of mixing water with the BTNA solution.

It's important to note that the specific reaction responsible for the color change cannot be determined without further testing. It is recommended to conduct additional experiments and analysis, such as characterization of the yellow product and testing for contaminants, to fully understand the reason for the color change.