Human RBCs: 15 mM Acetylphenylhydrazine treatment

When you start the program a window similar to Figure 1 will open. The first thing that you should do is to set the path to the directory containing your files.

For windows: If your files are in the directory "C:\spectra\june" then enter c:/spectra/june/
For linux: If your files are in the directory "spectra/june" located in your home directory then enter ~/spectra/june/
Note: you should use this / not \ and you must have / at the end of the file path.

Figure 1. Main window for winComp.

Main window buttons

Experiment Info

This button opens a window (Figure 2) where you may enter all the information that you wish to record about the experimental procedure and conditions. To save the information that you entered click the Save info button; by clicking this button you write the text that you have entered to a file named "info" in the directory that you specified as your path. In order to prevent overwriting previously generated "info" files the program will not write the text to the file if an "info" file already exists in the path directory (you have to manually delete the "info" file if you wish to save new text to that directory).

Figure 2. Experiment Info window.

Spectra Files

This button opens a window (Figure 3) where you will enter information about the files containing absorbance readings (you may use text or binary files, however, your files must be formatted and named according to rules that we will discuss below).

Filename format: Here we will use a generic filename and discuss what each component. Generic filename: PrefixTime00SpectraSuffix. In this filename there are four variable components:

Prefix: In this case we have named all our files with the prefix "aphz", so we must enter this as the prefix.
Time: For this example we took readings at 5 time points, so we enter 5 in the box under "# of Time Points".
Spectra: This is where you enter the number of spectra readings taken at each time point. In this example we had 3, so we enter 3 in the box under "# of Spectra per time pt".
Suffix: This is the suffix including the period. Since we are using binary files generated by the Beckman DU640, we have entered the suffix ".scn" in the box under "Suffix".

The 00 is invariable, we have incorporated it into the file name because of the manner in which the Beckman DU640 generates binary spectra files.

Figure 3. Spectra Files window.

Show Filename button: After entering your filename info, you can press this button and the filename, including the path, for the file (for the last file if you are using binary files) will be displayed. In this example, our path is "./aphz/" and the last filename is "aphz5003.scn" so "./aphz/aphz5003.scn" is displayed.

File Type: There are three different input file types supported:

Binary: Little endian If your spectrophotometer generates binary files for the absorbance readings in the same endian format as your computer reads them select this one.
Binary: Big Endian If your spectrophotometer generates binary files for the absorbance readings a different endian format than your computer select this one.
Text: Delimited If you have entered all of your absorbance readings into a text space-delimited (and maybe tab-delimited) file then select this one.

Text file format: You will need a text file with all of the data. When you generate your text file you must have the data entered in a specific order. Example: We took absorbance readings with a wavelangth range of 380-700 nm for 3 samples over 5 time points. So the first column should contain the numbers from 380 to 700. The second column should contain the absorbance readings for the first sample from the first time point, the third column should contain the absorbance readings for the second sample from the first time point, the fourth column should contain the absorbance readings for the third sample from the first time point, the fifth column should contain the absorbance readings for the first sample from the second time point, and so on.

If you are not sure if you entered the filename and file type information correctly, then press the "Plot Raw Spectra" button

Plot Raw Spectra button: If you have entered all the the file name parameters correctly and selected the correct file type then pressing this button with generate a series of plot windows similar to Figure 5. You will have one window for each time point and each window will have one plot for each sample. These plots are zoomable (left click and drag you box to select zoom area, right click to unzoom). If your spectra plots are empty or absurd, then you may have selected the wrong file type or entered the data into the text file incorrectly; try using a different file type and checking your file and path name (if it still does not work email the program support with a detailed explanation of the problem and a set of data files with a detailed explanation, if possible include binary and text files). If an error box is displayed press enter on your keyboard and verify that the filename and path are correct, then make sure that the data files are in the directory specified.

Figure 4. Example plot of the raw spectra.

Animal Species

Select human or bovine. Here we used human blood.

Globin

Select Hb for hemoglobin and Mb for myoglobin (Mb is currently not supported)

Heme Species

Select the heme species. For the competition experiment the only detectable species produced in the extracellular space are oxyHb and metHb.

Run Deconvolution

Now that you have entered the path, filename, and global paramters correctly you are ready to find out how much of each selected heme species is present in each sample for each time point. Pressing this button will pop up a window (Figure 5) containg timcourse plots of heme species for each sample and write a file name "hbconc" with the concentrations to the directory specified in your path (it will overwrite any preexisting file with the same name)

Figure 5. Time course plot of changes in heme species for each sample.

Show Residuals button: Pressing this button will pop up a series of windows (Figure 6; 1 for each time point) with a set of plots (1 for each sampl) these plots represent the difference between the raw spectra and the fitted spectra. If you look at Figure 4 you will see that the maximum difference between the raw spectra and the fit is ~1%.

Figure 6. Example plot of the raw spectra minus the fit used to determine the heme species concentration.

Enter Parameters and Run Comp Assay

Before you press this button you should have pressed the "Run Deconvolution" button because you need the values generated by the "Run Deconvolution" button to calculate the relative rates.

Pressing this button opens a window (Figure 7) where you enter the required experimental information. There are eight boxes to be filled in; the first 6 must have the same number of elements. Example: We treated red blood cells (RBC) with 15 mM acetylphenyl hydrazine (APZ) and wanted to compare the relative NO uptake rate with that of untreated red blood cells. Our first sample contained the untreated RBCs, extracellular hemoglobin (exHb), and NO donor. Our second sample contained APHZ-treated RBCs, exHb, and NO donor. Our third sample contained hemoglobin (Hb) and NO donor.

Box 1: Because we are interested in the rate that the RBCs uptake NO, we only enter the numbers for the RBC-containing samples here (1 2).
Box 2: Because we are interested in the NO uptake rate of RBCs relative to the reaction rate of NO with Hb (which was sample 3), we enter 3 3. Remember the 1st 6 boxes must have the same number of elements.
Box 3: This box is where you indicate what samples are going to be used to account for lysis. When you run the competition experiment you can have a control sample with just treated or untreated RBCs for each test sample or you can just enter the number of the test sample. Here we did not have additional controls so we enter 1 2. The lysis coefficient calculated using the test samples is usually not very different from a control sample.
Box 4: Here you enter the sample numbers to calculate the rate of autooxidation. You can have a Hb control and enter it here or use the Hb with NO sample number. Because autooxidation of Hb in the course of this experiment is not excessive we can just use the Hb with NO sample number so we enter 3 3.
Box 5: Here we enter the hematocrit for each sample entered in the first box. The hematocrit for sample 1 was 0.1 and for sample 2 was 0.12.
Box 6: Here we enter the internal metHb concentration for each sample in box 1. The internal metHb concentration for sample 1 was 0.04 and for sample 2 was 0.05. If you are using fresh untreated RBCs the internal metHb concentration is usually about 0.04.
Box 7: Enter the number of samples in Box 1.
Box 8: Enter the number of time points.

Figure 7. Window where you will enter the parameters for the competition assay.
Run Comp Assay button: Once you have entered all of the information correctlly, press this button. This button will pop up a window (Figure 8) that contains the results of the rate analysis. In the upper left hand side is a text box with some numbers; these numbers are the relative rates of reaction for NO with RBCs versus NO with free Hb. The first number corresponds to the first sample, the second to the second sample, ... . The plots are shown to verify that there is a linear relationship that increases to the right; if the plots are not linear then either the parameters were entered incorrecly or there was a problem with the experimentalist's technique (the Competition Assay requires practice to perfect, but is relatively simple once you have practiced enough). Sometimes in the course of an experiment one data point will be out of line, this is most often due to sample handling. If you have 4 or more data points then you can remove this "bad" point and recalculate the rate by pressing the "Drop Points" button; this button pops up a window where you select which points to drop for each sample (Note: even though this function calculates the rate correctly, as far as we know, after dropping the point the plotting function does not always work correctly).
Also, clicking the "Run Comp Assay" button writes the results to a file named "rates" in the directory you specified. Running the drop points function overwrites this file with the recalculated rates.

Figure 8. Results of the competition assay analysis