Access Count 
Porphyrin Analytical Tools
Porphyrin Analytical Tools (PAT) (Rebeiz and Saab, 1995) refers to a series of computer programs that allow the performance of quantitative and qualitative manipulations of tetrapyrrole spectral fluorescence data acquired in the Data.dat format using SLM software. To use this program the user has to load an SLM Data.dat file anywhere on his computer. If such a file is not available, email crebeiz@uiuc.edu. By return mail the user will reveive a zipped, self-extracting Data.dat file containing spectral data that allow the user to experiment with all the described PAT routines. The user should drag the Data.dat file attachment to any directory on the computer and accept extracting the file to that directory . In order to perform quantitative calculations with the Java routine, it is mandatory to download and install the Java (TM) 2 Runtime Environment Standard Edition file named: J2re-1_4_0_01-windows-i586. This self-extractable and -installable file can be downloaded from the following website: http://java.sun.com, by clicking the "Download J2seTMv1.4 Now" button. Once this file is installed click on the Porphyrin Analytical Tools link to access the calculation window. Following the appearance of the calculation window, a Java Security Warning Window is echoed to the screen. To activate the calculation window, click the Grant This Session button.
PAT programs are organized into three groups of modules namely an Access Module, Equation Module, and a Matrix Module. A brief explanation of each module is given below. A detailed description of the chemsitry and biochemistry undelying the described routines is given in an extensive review (Rebeiz, 2002).
Topics
Click on a topic to go directly to it.
- I. Access Module
- II. Equation Module
- 1. Proto
- 2. MPE amplitude or MPE area
- 3. Pide
- 4. Chla, Chlb, Pheoa, & b
- 5. DV Chl(ide) a
- 6. DV Chl(ide) b
- 6. 2CP MV, DV, MPE F591, F587
- 8. 2CP MV, DV, PIDE F625
- 9. 3CP MV, DV, Pide F625
- 10. 2CP MV, DV, Chl a F674
- 11. 2CP MV, DV, Chl b F666
- 12. MV Pide b
- 13. MV Pide Ester b
- III. The Matrix Module
- Example
- IV. Equations 26 to 51
- V. References
I. Access Module
The access module allows the user to access the recorded data of any acquired (i. e. recorded) SLM spectrum from within the PAT program. This module is used by the Equation and Matrix modules for all PAT calculations. The Access program is opened by selecting "identify spectra" from the selection window, and by entering a record number (#1-83) in the Rec# box. By clicking on the ClickHereToDisplayResults button, the spectrum properties are displayed in the display window. The user can print the screen to produce a hard copy of the displayed data. To use use this routine and identify the spectral parameters of a fluorescence spectrum stored in the Data.dat file spectrum, click on this Porphyrin Analytical Tools link.
II. Equation Module
This module consists of a series of programs that allow the quantitative determination of most of the metabolic pools of the porphyrin and chlorophyll biosynthetic pathways. When the PAT program is accessed, a series of equation titles appear in a selection window with a sliding bar. Each title corresponds to a set of equations that allow the quantitative determination of a particular tetrapyrrolel. The use of the various equations is described below:
1. Proto....E400, E440
This equation allows the quantitative determination of the amount of protoporphyrin IX (Proto) in the presence and absence of coproporphyrin, Mg-protoporphyrin IX, protochlorophyllide a and b and chlorophyllide or chlorophyll [chlorophyll(ide)] a and b from two fluorescence emission spectra recorded from 580 to 700nm in hexane-extracted acetone or 80% acetone, at room temperature. The two spectra are elicited by excitation at 400 and 440 nm respectively (Rebeiz et al, 1975; Rebeiz, 2002). When Proto....E400, E440 is selected, the user should enter the record number of the first emission spectrum (#1-83) elicited by excitation at 400 nm, in the E400 = box, and the number of the second emission spectrumd (#1-83) elicited by excitation at 440 nm, in the E440 = box. By clicking on the ClickHereToDisplayResults button, the amount of protoporphyrin IX in pmoles/ml of solution plus and minus standard deviation is echoed to the display window. The user can print the screen to produce a hard copy of the displayed data. To use use this routine and calculate the amounts of Proto from two emission spectra stored in the Data.dat file, click on this Porphyrin Analytical Tools link.
2. MPE area....E420
This equation allows the quantitative determination of the amount of Mg-protoporphyrin IX and/or Mg-protoporphyrin IX monomethyl ester [MP(E)] in the presence and absence of coproporphyrin, protoporphyrin IX, protochlorophyllide a and b and chlorophyll(ide) a and b, from one fluorescence emission spectrum, recorded from 580 to 700 nm at room temperature in hexane-extracted acetone, or 80% acetone, The emission spectrum is elicited by excitation at 420 (Smith and Rebeiz, 1977; Rebeiz, 2002). When MPE area ....E420 is selected, the user should enter the record number (#1-83) of the emission spectrum elicited by excitation at 420 nm, in he E420 = box. By clicking on the ClickHereToDisplayResults button, the amount of MP(E) in pmoles/ml of solution plus and minus standard deviation is echoed to the display window. The user can print the screen to produce a hard copy of the displayed data. To use use this routine and calculate the amounts of MP(E) from an emission spectrum stored in the Data.dat file, click on this Porphyrin Analytical Tools link.
3. Pide....E400, E440
This equation allows the quantitative determination of the amount of protochlorophyllide a (Pchlide a) in the presence and absence of coproporphyrin, Mg-protoporphyrin IX, protochlorophyllide b and chlorophyllide or chlorophyll [chlorophyll(ide)] a and b, from two fluorescence emission spectra recorded from 580 to 700 nm at room temperature, in hexane-extracted acetone, or in 80% acetone. The two spectra are elicited by excitation at 400 and 440 nm respectively (Rebeiz et al, 1975; Rebeiz, 2002). When Pide....E400, E440 is selected, the user should enter the record number of the first emission spectrum (#1-83) elicited by excitation at 400 nm, in the E400 = box, and the number of the second emission spectrumd (#1-83) elicited by excitation at 440 nm, in the E440 = box. By clicking on the ClickHereToDisplayResults button, the amount of Pchlide a in pmoles/ml of solution plus and minus standard deviation is echoed to the display window. The user can print the screen to produce a hard copy of the displayed data. To use use this routine and calculate the amounts of Pchlide a from two emission spectra stored in the Data.dat file, click on this Porphyrin Analytical Tools link.
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4. Chla, Chlb, Pheoa, & b....F674, F660
This set of equations allow the quantitative determination of the amounts of chlorophyll a and or chlorophyllide a [Chl(ide) a], chlorophyll and or chlorophyllide b [Chl(ide) b] pheophytin and or pheophorbide a [pheo(bide) a], pheophytin and or pheophorbide b [(pheo(bide) b] from two fluorescence excitation spectra recorded at room temperature, in hexane-extracted acetone or in 80% acetone, from 380 to 500 nm (Bazzaz and Rebeiz 1979; Rebeiz, 2002). When the Chla, Chlb, Pheoa & b...F674, F660 equation is selected, the user should enter the record number of the first excitation spectrum (#1-83) recorded at an emissiom wavelength of 674 nm, in the F674 = box, and the number of the second excitation spectrumd (#1-83) recorded at an emission spectrum of 660 nm, in the F660 = box. By clicking on the ClickHereToDisplayResults button, the amounts of Chl(ide) a, Chl(ide) b, Pheo(bide) a, and Pheo(bide) b, in pmoles/ml of solution plus and minus standard deviation are echoed to the display window. The user can print the screen to produce a hard copy of the displayed data. To use use this routine and calculate the amounts of Pchlide a from two emission spectra stored in the Data.dat file, click on this Porphyrin Analytical Tools link.
5. DV Chl(ide) a....F674, F660
This equation allows the quantitative determination of the amount of divinyl chlorophyll(ide) a [DV Chl(ide) a] from two fluorescence excitation spectra recorded at room temperature, in hexane-extracted acetone or in 80% acetone, from 380 to 500 nm ( Rebeiz, 2002). When the DV Chl(ide)a...F674, F660 equation is selected, the user should enter the record number of the first excitation spectrum (#1-83) recorded at an emissiom wavelength of 674 nm, in the F674 = box, and the number of the second excitation spectrumd (#1-83) recorded at an emission spectrum of 660 nm, in the F660 = box. By clicking on the ClickHereToDisplayResults button, the amount of DV Chl(ide) a, in pmoles/ml of solution plus and minus standard deviation is echoed to the display window. The user can print the screen to produce a hard copy of the displayed data. To use use this routine and calculate the amounts of DV Chl(ide) a from two emission spectra stored in the Data.dat file, click on this Porphyrin Analytical Tools link.
6. DV Chl(ide) b....F674, F660
This equation allows the quantitative determination of the amount of divinyl chlorophyll(ide) b [DV Chl(ide) b] from two fluorescence excitation spectra recorded at room temperature, in hexane-extracted acetone or in 80% acetone, from 380 to 500 nm ( Rebeiz, 2002). When the DV Chl(ide)b...F674, F660 equation is selected, the user should enter the record number of the first excitation spectrum (#1-83) recorded at an emissiom wavelength of 674 nm, in the F674 = box, and the number of the second excitation spectrumd (#1-83) recorded at an emission spectrum of 660 nm, in the F660 = box. By clicking on the ClickHereToDisplayResults button, the amount of DV Chl(ide) b, in pmoles/ml of solution plus and minus standard deviation is echoed to the display window. The user can print the screen to produce a hard copy of the displayed data. To use use this routine and calculate the amounts of DV Chl(ide) a from two emission spectra stored in the Data.dat file, click on this Porphyrin Analytical Tools link.
7. 2CP MV, DV, MPE... F591, F587 totalQ
This set of equations allows the quantitative determination of the amount of divinyl (DV) and monovinyl (MV) MP(E) in diethyl ether at 77 degree K, from two fluorescence excitation spectra recorded from 380 to 500 nm at emission wavelengths of 591 and 587 nm respectively (Tripathy and Rebeiz, 1985; Rebeiz, 2002). When the 2CP MV, DV, MPE... F591, F587 total Q equation is selected, the user should enter the record number of the first excitation spectrum (#1-83) recorded at an emissiom wavelength of 591 nm, in the F591 = box, and the number of the second excitation spectrumd (#1-83) recorded at an emission spectrum of 587 nm, in the F587 = box. The user should next enter the total amount of MV + DV MP(E) in the Total_Q box. By clicking on the ClickHereToDisplayResults button, the amounts of DV and MV MP(E), in pmoles/ml of solution plus and minus standard deviation is echoed to the display window. If the user enters a value of 100 in the Total_Q box, the percents of DV and MV MP(E), instead of the amounts of DV and MV MP(E) is echoed to the display window. The user can print the screen to produce a hard copy of the displayed data. To use use this routine and calculate the amounts of DV Chl(ide) a from two emission spectra stored in the Data.dat file, click on this Porphyrin Analytical Tools link.
8. 2CP MV, DV, PIDE... F625 total Q
This set of equations allows the quantitative determination of the amounts of divinyl (DV) and monovinyl (MV) Pchlide a and /or it sphytyl ester (Pchl(ide) a in diethyl ether at 77 degree K, from one fluorescence excitation spectrum recorded from 380 to 500 nm at an emission wavelength of 625 nm (Tripathy and Rebeiz, 1985; Rebeiz, 2002). When the 2CP MV, DV, Pide... F625, total Q equation is selected, the user should enter the record number of the excitation spectrum (#1-83) recorded at an emissiom wavelength of 625 nm, in the F625 = box. The user should next enter the total amount of MV + DV Pchl(ide) a in the Total_Q box. By clicking on the ClickHereToDisplayResults button, the amounts of DV and MV Pchl(ide) a, in pmoles/ml of solution plus and minus standard deviation is echoed to the display window. If the user enters a value of 100 in the Total_Q box, the percent of DV and MV Pchl(ide) a, instead of the amounts of DV and MV Pchl(ide) a is echoed to the display window. The user can print the screen to produce a hard copy of the displayed data. To use use this routine and calculate the amounts of DV Chl(ide) a from two emission spectra stored in the Data.dat file, click on this Porphyrin Analytical Tools link.
9. 3CP MV, DV, Pide F625
This set of equations allows the quantitative determination of the amounts of divinyl (DV) and monovinyl (MV) Pchlide a and /or it sphytyl ester (Pchl(ide) a in the epresence of MP(E) in diethyl ether at 77 degree K, from one fluorescence excitation spectrum recorded from 380 to 500 nm at an emission wavelength of 625 nm ( Tripathy and Rebeiz, 1985; Rebeiz, 2002). When the 3CP MV, DV, Pide... F625, total Q equation is selected, the user should enter the record number of the excitation spectrum (#1-83) recorded at an emissiom wavelength of 625 nm, in the F625 = box. The user should next enter the total amount of MV + DV Pchl(ide) a in the Total_Q box. By clicking on the ClickHereToDisplayResults button, the amounts of DV and MV Pchl(ide) a, in pmoles/ml of solution plus and minus standard deviation is echoed to the display window. If the user enters a value of 100 in the Total_Q box, the percent of DV and MV Pchl(ide) a, instead of the amounts of DV and MV Pchl(ide) a is echoed to the display window. The user can print the screen to produce a hard copy of the displayed data. To use use this routine and calculate the amounts of DV Chl(ide) a from two emission spectra stored in the Data.dat file, click on this Porphyrin Analytical Tools link.
10. 2CP MV, DV, Chl a.... F674 total Q
This set of equations allows the quantitative determination of the amounts of divinyl (DV) and monovinyl (MV) chlorophyll(ide) a in diethyl ether at 77 degree K, from one fluorescence excitation spectrum recorded from 380 to 500 nm at an emission wavelength of 674 nm (Wu et al, 1989; Rebeiz, 2002). When the 2CP MV, DV, CHLa... F674, total Q equation is selected, the user should enter the record number of the excitation spectrum (#1-83) recorded at an emissiom wavelength of 674 nm, in the F674 = box. The user should next enter the total amount of MV + DV Chl(ide) a in the Total_Q box. By clicking on the ClickHereToDisplayResults button, the amounts of DV and MV Chl(ide) a, in pmoles/ml of solution plus and minus standard deviation is echoed to the display window. If the user enters a value of 100 in the Total_Q box, the percent of DV and MV Chl(ide) a, instead of the amounts of DV and MV Chl(ide) a is echoed to the display window. The user can print the screen to produce a hard copy of the displayed data. To use use this routine and calculate the amounts of DV Chl(ide) a from two emission spectra stored in the Data.dat file, click on this Porphyrin Analytical Tools link.
11. 2CP MV, DV, Chl b... F666 total Q
This set of equations allows the quantitative determination of the amounts of divinyl (DV) and monovinyl (MV) chlorophyll(ide) b in diethyl ether at 77 degree K, from one fluorescence excitation spectrum recorded from 380 to 500 nm at an emission wavelength of 666 nm (Wu et al, 1989; Rebeiz, 2002). When the 2CP MV, DV, CHLb... F666, total Q equation is selected, the user should enter the record number of the excitation spectrum (#1-83) recorded at an emissiom wavelength of 666 nm, in the F666 = box. The user should next enter the total amount of MV + DV Chl(ide) b in the Total_Q box. By clicking on the ClickHereToDisplayResults button, the amounts of DV and MV Chl(ide) b, in pmoles/ml of solution plus and minus standard deviation is echoed to the display window. If the user enters a value of 100 in the Total_Q box, the percent of DV and MV Chl(ide) b, instead of the amounts of DV and MV Chl(ide) b is echoed to the display window. The user can print the screen to produce a hard copy of the displayed data. To use use this routine and calculate the amounts of DV Chl(ide) a from two emission spectra stored in the Data.dat file, click on this Porphyrin Analytical Tools link.
12. MV Pide b
This set of equations allows the quantitative determination of the amount of monovinyl (MV) Pchlide b in a mixture of Pchlide a and b diethyl ether at 77 degree K, from two fluorescence emission spectra elicited by excitation at 440 and 463 nm respectively. When the MV Pide b title is highlighted and entered, the user is prompted to enter the record number of the first emission spectrum recorded at an excitation wavelength of 463 nm. When the first record number is entered, the user is prompted to enter the record number of the second emission spectrum recorded at an excitation wavelength of 440 nm. When that record number is entered, the user is prompted to enter the total amount of Pchlide a in the mixture which is determined as described above for Pide. When the total amount of Pchlide a is entered, the amounts of MV Pchlide b plus and minus standard error is immediately echoed to the screen. If the amount of total Pchlide a had not been determined, the user can enter the arbitrary number of 100. In that case the equations give the percent of MV Pchlide b in the mixture. The user can print the screen to produce a hard copy of the calculated data. By pressing escape the user is reverted back to the main PAT screen.
13. MV Pide Ester b
This set of equations allows the quantitative determination of the amount of monovinyl (MV) Pchlide phytyl ester b (Pchlide E b) in a mixture of Pchlide E b and Chl a and b in diethyl ether at 77 degree K, from two fluorescence excitation spectra recorded at emission wavelengths of 643 nm and 660 nm respectively. When the MV Pide ester b title is highlighted and entered, the user is prompted to enter the record number of the first excitation spectrum recorded at an emission wavelength of 643 nm. When the first record number is entered, the user is prompted to enter the record number of the second excitation spectrum recorded at an emission wavelength of 660 nm. When that record number is entered, the user is prompted to enter the total amount of Chl b in the mixture which is determined as described above for Chla, Chlb, Pheoa & b. When the total amount of Chl b is entered, the amounts of MV Pchlide E b plus and minus standard error is immediately echoed to the screen. If the amount of total Chl b had not been determined, the user can enter the arbitrary number of 100. In that case the equations give the percent of MV Pchlide E b in the mixture. The user can print the screen to produce a hard copy of the calculated data. By pressing escape the user is reverted back to the main PAT screen.
III. The Matrix Module
This program determines the number of fluorescent compounds in a solution containing a mixture of fluorescent species (7). The program is based on Weber's matrix technique (8). Weber demonstrated that for an emission-excitation matrix "M" whose elements "Mi,j", represent the fluorescence intensity measured at wavelength "W j", for an excitation "W I", the number of fluorescent components depicted in the matrix is equal to the rank of the minors for which the following inequality is obeyed:
Delta/P = or > 3.GammaF/F' (Eq. 1)
where:
Delta = value of the determinant for a minor of any particular rank "N".
P = value of the permanent ( i. e. the sum of all possible diagonal products of the matrix).
GammaF: the noise level or minimum detectable fluorescence (in fluorescence units).
F': the mean fluorescence intensity of the Mi,j elements of the minor under consideration.
Example:
Let us assume that a user has on hand a solution in 80 % acetone consisting of a mixture of fluorescent compounds. Let us assume that the user suspects that the solution may contain as many as 6 fluorescent compounds. To determine the actual number of fluorescent compounds in the solution the user records 7 emission spectra elicited by excitation at 400 nm (record # 1), 410 nm (record # 2), 420 (record # 3), 430 nm (record # 4), 440 nm (record # 5), 450 nm (record # 6), 460 nm (record # 7). In order to determine whether the solution contains 6 fluorescent compounds the user tells the program to assemble a 7 X 7 matrix from records, 1-7, then (b) tells the program to determines all the minors of rank "6" which obey Eq. 1. For example if several minors of rank 6 are found which obey Eq. 1, it means that there are at least 6 fluorescent compounds in the mixture. If none of the minors are found to obey Eq. 1, the user would conclude that there are less than 6 fluorescent compounds in the mixture. He would then query the matrix for all 5 X 5 minors that obey Eq. 1 etc...
IV. Equations 26 to 51
Equations 26 to 51 are user defined. They can be programmed by the user.
V. References:
- Rebeiz, C. A., and Daniell G. Saab (1995). Porphyrin Analytical Tools (Copyrighted software).
- Rebeiz, C. A., B. B. Smith, J. R. Mattheis, C. C. Rebeiz and D. F. Dayton (1975). Chloroplast biogenesis. Biosynthesis and accumulation of protochlorophyll by isolated etioplasts and developing chloroplasts. Arch. Biochem. Biophys. 171: 549-567.
- Smith, B. B. and Rebeiz, C. A. (1977). Spectrofluorometric determination of Mg-protoporphyrin monoester and longer wavelength metalloporphyrins in the presence of Zn-protoporphyrin. Photochem. Photobiol. 26: 527-532
- Bazzaz, M. B. and C. A. Rebeiz (1979). Chloroplast culture V. Spectrofluorometric determination of chlorophyll(ide) a and b and pheophytin (or pheophorbide) a and b in unsegregated pigment mixtures. Photochem. Photobiol. 30: 709-721
- Tripathy, B. and C. A. Rebeiz (1985). Chloroplast biogenesis: Quantitative determination of monovinyl and divinyl Mg protoporphyrins and protochlorophyll(ides) by spectrofluorometry. Anal. Biochem. 149: 43-61.
- Wu, S. M., Mayasich, J. M. and Rebeiz, C. A. (1989) Chloroplast Biogenesis: Quantitative determination of monovinyl and divinyl Chlorophyll(ide) a and b by spectrofluorometry. Anal. Biochem. 178: 294-300
- Belanger, Faith C. and C. A. Rebeiz (1980). Chloroplast Biogenesis. Detection of divinyl protochlorophyllide in higher plants. J. Biol. Chem. 255: 1266-1272
- Weber, G. (1961) Nature. 190: 27-29.
- Rebeiz, C. A. (2002). Analysis of Intermediates and end products of the chlorophyll biosynthetic pathway. In Heme, Chlorophyll, and Bilins: Methods and Protocols, (A. G. Smith, and M. Witty eds.) Humana press , pp 111-155
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