Have you checked the pH of the original 1X buffer and the diluted 10X buffer to see if they are same?

I'm not clear on the reason you need to increase the buffer concentration to compensate for increased ethanol concentration. Also, have you considered that the ethanol concentration may also affect enzyme activity or absorbance?

Alk.ph enzyme activity is dependent on pH, and the substrate concentration not much on other factors. You need to keep standards absorption range comparable with the test samples. Lower concentrations of buffer salts are preferred . However 10x diluted to 1x should not give different results. One should have all reagents in the same and constant while changing only the standard concentration as multiples to have a linear graph.

No need to change the buffer concentration or the volume of the cocktail

The absorbance of a glycine buffer depends on the zwitterion that exists in solution on both parts of the isoelectric point, a pH value that depends on the glycine concentration. This means that you must adjust the pH of your concentrated solution at a value that is very close to that of the buffer in which you will do your measurements/experiments.

Sincerely

CC

Apologies for the lack of clarity guys and thank you for the responses :)

The normal protocol for the assay I'm using calls for 50 uL of substrate(pNPP) + 50 uL of buffer (0.1M glycine buffer) with 10 uL of enzyme. In the experiment testing ethanol's effect, I need to increase the buffer to 10x because I am diluting down 10-fold by the addition of Ethanol and dH20 into the buffer mix. So for example:

5% Ethanol experiemnt: 50 uL of S. + 50 uL of buffer mix (10uL of 10x Buffer + 11 dH20 + 79 uL EtOH) this will give an EtOH concentration of 5% after dilution with substrate and enzyme. 

I have made both buffers up to pH 8.8 as per method. 

The regular buffer is giving absorbance around 0.5 - 0.8, but the 10x buffer (diluted down to 1x) is giving absorbance of >1.0...it's just very odd.

Thank you again.

My only question: what is the action of EtOH on glycine molecules? I cannot answer this question.

CC

CC: I cannot find any evidence of EtOH and glycine interactions. 

The main issue is that the rise in absorbance is being seen with AND without ethanol, which would suggest to me problems with the buffer, perhaps even dilution problems, but I am very confident against the latter.

There is some confusion with your protocol:

"5% Ethanol experiment: 50 uL of S. + 50 uL of buffer mix (10uL of 10x Buffer + 11 dH20 + 79 uL EtOH) this will give an EtOH concentration of 5% after dilution with substrate and enzyme."

Is the volume of the buffer mix 50 µl or 100 µl, and what was the volume of ethanol added? The final ethanol concentration is surely higher than 5%, in either case. A high concentration of organic solvent could affect both the enzyme activity (probably reducing it), and the extinction coefficient of the product.

Glycine itself doesn't have any visible absorbance.

I know that the following paper is old, but:"the best broths are cooked in the oldest pans"… even if spectroscopy has evolved considerably…

THE ABSORPTION SPECTRA OF GLYCINE SOLUTIONS

AND THEIR INTERPRETATION

GLADYS A. ANSLOW, MARY LOUISE FOSTER, AND

CHARLOTTE KLINGLER

(From the Departments of Physics and of Chemistry, Smith College,

Northampton)

(Received for publication, July 19, 1933)

As stated above, it is possible that the interaction between all the molecules included in solution could induce considerable changes in the organisation of the solution itself, inducing a large modifications of absorbance…

CC

In Google Books this blurb suggests Absorbace varies with concentratuion of Glycine:  

The Laboratory Digest - Volumes 37-38 - Page 12

https://books.google.com/books?id=aCfuAAAAMAAJ

1973 - ‎Snippet view - ‎More editions

Introduce either 2 ml of urine, 4 ml of protein-free supernatant fluid, or 4 ml of each ofglycine working standards into the column and ... Construct a concentration vs. absorbancecurve and calculate the concentration of glycine in the samples.

Hi Philip, I am confused.

I did lots of alkaline phosphate activity measurements in a previous life, but I never used ethanol in any of the reagents.

Why are you using ethanol in your alk. phos. assays?

EtOH decreases the solubility of many salts, so the increase in Abs. might be due to undissolved salt particles.

Hi Steingrimur,

I am using ethanol to destable the enzyme because my research is to show the stabilising effect of sugars (xylose).

My problem is not to do with the Ethanol per sé. As i am introducing ethanol into the reaction, I need to use a 10x glycine buffer, as the buffer becomes diluted as i add the Ethanol.

However, I used the 10x buffer diluted to the 1x concentration needed for the assay in a supporting experiment without any ethanol. But the absorbance was much higher than before.

Kind regards,

P.