I am trying to determine pyruvate kinase activity from crucian carp (Carassius carassius) muscle and liver. I am working on a photometric assay, where the formation of pyruvate and ATP from PEP and ADP by pyruvate kinase is coupled with the formation of lactate and NAD+ from pyruvate and NADH by LDH. The concentration of NADH is measured photometrically at 340 nm. As NADH is oxidized the absorption should decrease. However, in most of my tests, it has increased. I have checked all my reagents and have no idea what is causing this and how to fix it. Has anyone used this assay (and had similar problems)?
The incubation has been done at 25˚C, and the tissue samples have been homogenized in 20X volume of triethanolamine buffer (0,1M, pH=7,6). The concentrations in the test solution are the following:
- Triethanolamin 85,6mM
- PEP 0,54mM
- MgSO4 2,5mM
- KCl 10mM
- ADP (neutralized with KOH) 4,7mM
- NADH 0,2mM
Dear Joonas,
You can use two common procedures for determination of PK activity. In both methods, at first you have to convert PEP + ADP to Pyruvate and ATP. Then Pyruvate in first method must be substrate of Pyruvate oxidase or in second method, substrate of LDH.
Pyruvate oxidase catalyzes the conversion of pyruvate to acetyl phosphate, hydrogen peroxide (H2O2), and carbon dioxide. You can determine hydrogen peroxide with conjunction of HRP in a dozen colorimetric, flurometric and luminometric assay.
LDH convert pyruvate + NADH to Lactate and NAD+ and you can follow reduction of absorbance at 340nm.
I think your problem in second method related to reagent concentration.
e.g. ADP in this method must be around 50mM and you used 1:10 dilution
e.g. PEP in this method must be around 50mM and you used 1:100 dilution
e.g. NADH in this method around 5mM and you used 1:25 dilution
you can repeat the assay with worthington procedure:
0.05 M Imidazole⋅HCl buffer, pH 7.6 2.7 ml
45 mM Adenosine diphosphate 0.1 ml
6.6 mM NADH 0.1 ml
45 mM Phosphoenolpyruvate 0.1 ml
Lactate dehydrogenase 0.01 ml
http://www.worthington-biochem.com/pkl/assay.html
best
mehdi
Joonas,
There is always a potential problem with the oxidation of NADH due to age and storage conditions. You should be able to break your reactions into 2 separate reactions to troubleshoot your assay. First, take advantage of the reversability of LDH and assay for the conversion of lactate and NAD to pyruvate and NADH. NADH is a chromafluor (ex 340/em 465). If this reaction works, you can test your NADH with pyruvate, using LDH as catalyst, and measure reduction of fluorescence (loss of NADH) or absorption gain at 340 (formation of NAD). The thermodynamics of the pyruvate kinase reaction makes it want to proceed in one direction, but once you know that your NADH is good, it should be straight forward to troubleshoot that reaction as well.
Hope this helps,
Dave
Dear Joonas,
Have you ever tried the assay with your sample but without ADP? The assay reaction do not work without ADP, however NADH absorption may be change (increase or decrease) for few minutes. We think these A340 change is background reaction. When background reaction is stable, you can add ADP in the reaction mix, then start measurement of PK activity depend on ADP. In this check method, ADP can be substituted by PEP.
Good luck!,
Daisuke
hello Joonas,
I have not used your technique, but I had a similar problem with a similar reaction using both NADH / NAD: absorbance increased instead of decreasing and only on some samples! I realized that slight turbidity appeared during the reaction. I was forced to abandon the technique.
The problem may be a molecule of the tissue extract you analyze
Why not use a commercial kit? MAK072 Sigma Aldrich. These products are generally very reliable
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