Standard Operating Procedure for the Cary 50 – Kinetics/Enzyme Kinetics – NAD+/NADH
Things to know:
1) The stability of NADH solutions is dependent on the buffer used.
[SB1]A useful paper to look at would:
Rover, L. "Study of NADH Stability Using Ultraviolet–Visible Spectrophotometric Analysis and Factorial Design." Analytical Biochemistry 260.1 (1998): 50-55.[SB2]
2) The generally accepted extinction coefficient for NADH is 6220[SB3]. A calibration curve was done for this machine and that data can be found in Figure 1 and Table 1 below.
As you can see the extinction coefficient is not the expected 6220, which is fine[SB4], and the literature value can be used just know that there will be some systematic error in the data if 6220 is used.
You can also take a look at The Extinction Coefficients of the Reduced Band of Pyridine Nucleotides which is a paper by Horecker and Kornberg published in 1948.
3) The Cary 50 and the plate reader have difficulties reading very low concentrations of NADH. This can be seen when comparing no NADH to those values with less than 0.01[SB5]mM NADH present.
Preparing the sample:
1)Prepare the stock solutions[SB6] if you have not already done so.
2)Locate the 1 mLcuvettes[SB7].
3)Pipette the required amount of substrate(s), cofactor and buffer solution into the cuvette. Withhold the enzyme until last and add this only after the Cary 50 has been set up for that particular sample.
Using the machine:
Running Samples
Choose the a[SB8]ppropriate software to use. There are two different programs that the Cary-50 offers that can be used to collect enzyme kinetics data, Enzyme Kinetics and Kinetics, the enzyme kinetics software can be easier to use, in that the software itself can give an initial velocity, however it will not give you an error estimate on that initial velocity.
Once you have chosen, read through the setups below for that specific software.
Open the sliding door of the machine.
Place the cuvette with the sample inside the machine making sure that one of the translucent two sides, face toward you.
For the UV transparent disposable cuvettes there is a triangle on one side of the cuvette make sure that is facing the detector.
Now press the Start button on the screen. (Top, center of screen)This should bring up a save menu.
Pick a name for your sample and save it. Press OK. This should bring up another screen. Press OK. Now you have two minutes to put the enzyme in the cuvette.
Take your pipette filled with enzyme and place into the cuvette in the machine. Release the enzyme from the pipette and then mix by pumping the pipette 5-10 times. This should be done relatively quickly.
Remove the pipette tip from the solution and release as much of the solution into the cuvette as possible.
Close the sliding door and Press OK on the computer screen.
The machine is now collecting data. To zoom in on the area you would like to view.
Right click on the screen and choose Axes Scales. Change the y-axis to around where you believe your sample is and press OK.
After the machine is done reading, remove the sample and insert the next one, following the same steps, or close out of the program and clean up your area.
Enzyme Kinetics
Open Enzyme Kinetics software – it should be in Start, most used programs box, but if not.
Start → All Programs → Cary WinUV → Enzyme Kinetics
Click the Setup icon (Upper left of the screen)
Cary – Here you can change:
Wavelength (default 500nm)
The wavelength for NADH is 340 nm.
Average Time (how often it reads)
Absorbance minimum and maximum
How long you want to run the sample for (default 1min)
Options
Decide if you want it displayed individually or as overlay
Accessories
Temperature Stuff (Have not used it)
Analyze
Calculate Vo
- input start and stop times (min)
- productabsorptivity (µM/cm) (extinction coefficient)
- path length (default 1 cm, standard cuvette)
Determine what you want to analyze with
- linear least squares, or
- Marquardt
Model
- non competitive
- competitive
- uncompetitive
Plot/Fit
- Lineweaver-Burk (default)
- Eadie-Hofstee
- Hanes-Woolf
- Eadie-Scatchard
- Vo vs. [S]
- Dixon 1/Vo vs [I]
Samples
Put in the number of samples
Name them
Put in the substrate (cofactor) concentration you are changing in (µM)
Put in the inhibitor concentration you are changing in (µM)
Reports
Various options for what will be included in the report section
Autostore (never used it)
Click Ok.
Now you can start collection (Press Start)
- Make sure you run the samples in the order that you named them in the samples section
After Collecting Data
- Will output all the trials run without a Vo
- In order to obtain the Vo click on Recalculate in the bottom left corner
- This will take you to the Analyze page (above)
- Click Ok
- Outputs the Vo along with the concentration values inputted earlier
**Note** It is possible to determine the Vo for individual files that are .BKN however this can only be done one at a time and will not display in the Kinetics Report area
- Open the file – Click on Graph (in the tool bar with File, Edit, etc.) – Click User Data Form… - Input values (For Vo just put in 0) – Click Ok
- Click Recalculate – Put in the Start and Stop times and Product Absorptivity (Extinction coefficient) – Click Ok
- A Error box will appear, the says ‘There is not enough data to evaluate’ (Ignore it) – Press Ok – Go back to the User Data Form – The Vo for your given parameters will be displayed under the Vo section
Converting Kinetics .BKN to Enzyme Kinetics files .BEK
- Open Kinetics .BKN file in either the Kinetics or Enzyme Kinetics programs (in Enzyme Kinetics you will have to choose All Files in order to view the .BKN files)
- File – Save As - .DEK
Once you have done this with all the files want to analyze with the EK program
- Open the EK program
- File – Open Data files – Click the Overlay check box – Highlight all files you want opened
- Click Open
- Will put the data onto one graph
- Click on the Trace Preferences symbol, it is right next to the Setup button
- Change the trace names so that they are recognizable when they are outputted – Right click on the name – Select Change trace name – Change the name – Click Ok.
- Click Ok after you have changed all the names
- Now go to Graph (it is in the tool bar with File, Edit, etc)
- Select User Data Form..
- Initially have Vo be 0 and put in the appropriate concentrations for S, and I
- Click on Recalculate
- Input the correct Start and Stop and the extinction coefficient
- Click Ok.
- The initial velocities should be outputted in the Report area below the graph
- To get a copy of the report click Print…
- Click Ok.
- Now save the file to a folder – this file will be able to be opened by internet explorer
Kinetics
Start up the program labeled Kinetics
Start → All Programs → Cary WinUV → Kinetics
Click the Setup icon (Upper left of the screen)
Cary – Here you can change:
Wavelength (default 500nm)
The wavelength for NADH is 340 nm.
Average Time (how often it reads)
Absorbance minimum and maximum
How long you want to run the sample for (default 1min)
Converting the data:
After the data has been collected using the Kinetics program it needs to be converted to a useable format.
Go to where you have saved your data and open one of the files.
Click on File | Save Data As | type in the file name. Below where you enter the filename there is the drop down bar for choosing a file type. Choose spreadsheet.
Press Save. (Your file can now be opened in Excel)
Repeat for the remainder of the samples.
The following Table 1 and Figure 1 show the calibration data for NADH in the Cary 50. Figures 2 and 3 show data collected for the enzyme malate dehydrogenase. This data was collected using both the Kinetics and Enzyme Kinetics software, but was analyzed entirely with the Enzyme Kinetics software.[SB9]
Table 1. NADH in PBS calibration data collected using the Cary 50
Concentration of NADH (mol/L) / 0 / 0.000002 / 0.00001 / 0.0001 / 0.0002 / 0.0003Absorbance / 0.137249 / 0.148105 / 0.180713 / 0.622172 / 1.064097 / 1.593927
Figure 1. Concentration of NADH verses absorbance in order to obtain the extinction coefficient, data points are from Table 1.
Figure[SB10] 2. Initial velocities verses the substrate concentrations at a malate dehydrogenase concentration of 0.02µM , (A) [NAD+] 0.1, 0.2, 0.5, 1, 5mM, (B) [Malate] 2, 2.5 ,3.3, 5, 10, 50mM. Reactions take place in a 50mM phosphate buffer solution at pH 7.0.
Figure 3. Initial velocities verses the substrate concentrations at a malate dehydrogenase concentration of 0.002µM , (A) [NADH] 0.015, 0.0375, 0.06, 0.18, 0.3mM, (B) [OAA] 0.01, 0.03 ,0.05, 0.15, 0.25mM. Reactions take place in a 50mM phosphate buffer solution at pH 7.0.
[SB1]Don' use excessive tabs. If yuo need to, just indent the line using the "Increase Indent" button or some equivalent
[SB2]Can we use Endnote or Mendeley for these?
[SB3]At what wavelength?
[SB4]Can you explain why?
[SB5]space between value and unit
[SB6]describe how to do this and concentrations
[SB7]Give a list of materials at the top.
[SB8]Probably should number these steps
[SB9]Give the conditions (pH, temperature, enzyme concentration)
[SB10]Probably should add representative absorption-time curves.