# Spectrophotometric assay by pNPP

Here is a series of related questions that I want to ask. If you think they ought to be different questions, please tell me so.

Background

The activity of acid phosphatase is measured by an enzymatic reaction that converts pnitrophenyl phosphate (pNPP) to p-nitrophenol (pNP), liberating phosphate. The product, pNP, absorbs light whose wavelength is 400 nm with an absorption coefficient( 400 nm) of 19000 M-1 cm-1 at extremely alkaline pH. Reaction mixture for an acid phosphatase is slightly acidic. Thus, it must be alkalinized for quantification of pNP.

Two enzyme concentrations are to be examined - They are 1X and 0.1X (The 0.1X enzyme is made by mixing 1ml of 1X enzyme in 9ml of NaCl.) Reaction times for each enzyme is 1,10 and 20 minutes.

Procedure

Protocol for measurement of acid phosphatase activity

１）Mix 0.12 ml of 0.5 M Na acetate buffer (pH 5.6) and 0.24 ml of 5 mM pNPP in a test tube. Start the reaction by adding 0.24 ml of an enzyme solution.

２）After the reaction times of 1, 10, and 20 min, respectively, stop the reaction by adding 0.6 ml of 0.5 M NaOH. NaOH stops the reaction and converts the pNP produced into a yellow-colored (A400-absorbing) form.

３）After all reactions are stopped, measure A400 of the samples There are some things I am supposed to calculate after I obtain the results.

First I am going to plot a graph of absorbance versus time and find the slope for each of the line.( 1X and 0.1X)

Now I have been asked to find absorbance change/min/1ml of 1X enzyme.

My answer : Divide the slope obtained by 0.24(the amount of enzyme)

Next I have to convert absorbance change to concentration change when L is 1cm and e400 of pNP is 19000/M/cm.

My answer : This can be found out by A = eCL

The next question is to convert the concentration change to a change in a number of moles of pNP.

My answer : I have no clue how to do this !

Finally, I have to calculate total activity (in moles per minute) in 4ml of 1X enzyme solution.

My answer : Multiply the answer I obtain in the above question by 4.

Thanks for surviving it till here :)

For the first subquestion:

Using unit analysis, the answer is very quickly found: The unit of the slope $m$, assuming dimensionless values for the absorbance, is $$[m] = \frac{[\Delta y]}{[\Delta x]} = \frac{[\Delta A]}{[\Delta t]} = \frac{1}{\text{min}}$$

Now you need a volume in the denominator as well, so dividing by a volume seems like a good idea. Since you need the change in absorption per mL of 1X solution, you divide the slope of the 1X reaction by 0.24 mL for the correct value.

For the subquestion with which you seem to have the most trouble: Simply apply $c=n/V$.

You will find the number of moles changed via $$\Delta n = \Delta c\times V = \Delta c \times 1.2~\mathrm{mL}$$

• Hi! If we have to find change in moles of pNP shouldn't we multiply it by 0.24mL (instead of 1.2mL) ? – biogirl Feb 28 '14 at 6:16
• No, because you measure the concentration of pNP in the 1.2 mL solution. – tschoppi Feb 28 '14 at 8:11
• So was I correct when I divided the slope by 0.24 ?( see the subquestion 1 ) – biogirl Mar 1 '14 at 8:12
• Since you don't give me the graph that you obtained it is really hard to comment on. Is the process linear in time? – tschoppi Mar 1 '14 at 12:48
• Yes! It is linear. – biogirl Mar 1 '14 at 15:27