General
PLEASE READ THE README FILE (CHELREAD.ME) ON THE DISKETTE!
CHELATOR is intended to be freeware (public domain). You may freely
copy and distribute it, but I urge you to include all the files (or,
as a failsafe alternative, copy only PACKCHEL.EXE and run it).
Although I've done my best to include only well-established stability
constants and double-checked all constants in the default datafile, I
cannot guarantee that what this program calculates is the absolute
truth under all imaginable conditions. A fact is that the default
datafile primarily contains constants measured near 37 degrees C. In
such a way I do not have to extrapolate constants determined at 25
degrees C too much for my own experiments. If you mainly perform
experiments at 20 degrees C or so, you are probably better off
changing the stability constants in your personal datafile (more on
this later).
At run-time, you need CHELATOR.EXE and your datafile .DAT (default:
CHELDFLT.DAT) in the current directory.
An introductory explanation about the program may be obtained by
typing a question mark after the program name (separated by a space)
at the DOS prompt. In order to obtain the requested information, the
CHELATOR.TXT file must be present in the current directory.
The terms 'metal' and 'chelator' should be self-explanatory to the
users of this program. The term '(pH )buffer' might need some
elaboration: if a compound acts as a pH buffer, BUT BINDS METAL IONS
AS WELL, it should be entered as a 'chelator'. The H+ binding action
of such compounds will be accounted for when stability constants for
binding of H+ have been entered (which, by the way, is obligatory!).
Only when a compound has no known affinity for the metal ions in the
system should it be entered as 'pH buffer'.
NOTE: CHELATOR was implemented in Borland Turbo Pascal 5.5.
Introduction
CHELATOR is a menu-driven calculation program written in Turbo Pascal
5.5þ for use on IBM-PC compatible computers.
CHELATOR is a greatly enhanced version of a previous program for
calculating free metal cation concentrations based on the method used
by van Heeswijk, Geertsen and van Os, J. Membrane Biol. 79, 19-31
(1984). Some errors in this routine have now been corrected. Also,
ten other possilbe equilibria can be considered per metal-chelator
pair. We reported on this program in Biotechniques.
The program automatically recalculates metal-chelator stability
constants for effects of ionic strength, temperature, and pH. All ion
activities are converted to their respective concentrations using the
activity coefficient derived for the ionic strength. Where possible,
temperature effects are calculated by Van 't Hoff's Isochore. A
detailed description of the methods used can be found in
Biotechniques.
Menu's
CHELATOR uses a menu-based structure much like that of popular
programs such as PC SHELLþ6 or Borland Turbo Pascalþ5.5.
The menus may be operated by cursor keys or a Microsoftþ
compatible mouse. When you use a mouse, please remember that the left
button is confirmative (ENTER), while the right button cancels (ESC).
You may drag the menus (i.e. moving through the menus with the left
mouse button pressed), but double-clicking is not supported (and not
necessary). Note that dragging is not supported in any of the dialog
boxes! Dialog boxes only support cursor keys, or simple mouse
clicks.
When you use CHELATOR, you generally should proceed through the main
menu from left to right.
File
The option 'Load' in the 'File' menu will give you a listing of
.DAT files in the CURRENT directory from which you may pick one file.
The datafile will be read. This file contains information on metals,
chelators, stability constants, pH buffers, media composition and
special program options. Once you are familiar with the program you
can save your personal named datafile with the 'Save' option in the
'File' menu. It is not allowed to overwrite the default datafile. The
'Edit' option further expands this flexibility.
Edit
The 'Edit' option of the main menu should NOT be used until you
are thoroughly familiar with CHELATOR. You normally skip this menu
item. It serves to configure datafiles. You may add, delete or change
metals, chelators, pH buffers, or specify stability constants
regarding a specific combination of metals and chelators. In this
way, you may formulate a datafile containing only those data you are
concerned with. Please be familiar with the theoretic background of
stability constants before changing any constants!
The maximum number of entries for chelators, metals and pH-buffers is
15. You may freely delete entries you will never use from your
personal datafile. However, CHELATOR will not permit you to delete
H+. Also, you MUST enter K1 and K2 for binding of H+ when you add a
new chelator to the list.
When you add a chelator, you are asked for its name (maximally 5
characters long), the stability constant for self-association (C + C
<-> C2), and its valence (usually negative). Then, the program
proceeds through the list of available metals to allow you to enter
stability constants for several equilibria. You must add the first
two constants K1 and K2 for H+. If you do not do this, 'Prepare
calculations' will abort with an error. You proceed to the next metal
by pressing F9 (Accept) or clicking the left mouse button when the
mouse cursor is on the Accept-field.
Metals are added in a similar way. You are asked to enter a name
(maximally 4 characters; I suggest you use 'Xx?+', where 'Xx' is the
commonly used mnemonic for this metal and '?' is its valence). Next,
CHELATOR asks for the valence of the metal. After this, the program
proceeds through the list of chelators in order to let you provide
stability constants.
When adding a pH-buffer to the datafile, one must enter its name (up
to 15 characters), its kind (see below), its pKa at 25 degrees C, and
its delta pKa at 25 degrees C. We describe the kind by two terms:
Cationic is used when the pH-buffer is positively charged at
pH<pKa, and anionic when the pH-buffer is not charged at
pH<pKa.
Please remember that changes made in the datafile are only made in
the computer's memory. They are not saved automatically! You must use
'Save' in the 'File' menu to save the modified datafile to disk.
Media
The 'Media' option of the main menu is used to inform the program
about the composition of your assay media and the experimental
conditions. Here you may enter which chelators, metals and pH buffers
you choose to use in your experiment. The program will ask for the
total concentrations of the chelators and pH buffers. Furthermore,
you are asked to enter pH, temperature and final ionic strength of
your media.
The latter option requires some explanation: the figure is entered in
ionic equivalents.
Total ionic strength = summation(c*|z|)/2;
where c = concentration and z = valence of anion or cation. So
CHELATOR wants you to add up concentration times valence, not
concentration times valence2, as in 'classical' ionic strength. The
figure you enter in 'ionic strength' represents the total ionic
strength of the media. The program then computes the contribution to
ionic strength of pH buffers, free chelators, free metals and all
complexes. In calculating the contribution to ionic strength of the
added metal salts involved in the calculation, the program assumes
that these salts have MONOVALENT counter-anions!!
The program calculates the degree of ionization of pH buffers by
(re)calculating their pKa's as a function of temperature (according
to Good et al. Biochemistry 5: 467-477 (1966), correction for ionic
strength in the physiological range is semi-absent), and thereby the
buffers' contribution to the final ionic strength.
When calculating free metal cation concentrations, the program will
subtract contributions of complexes, free metal cations, ionized pH
buffers, etc. from the figure of 'total ionic strength' and will end
up with a 'medium ionic strength', representing the actual ionic
strength of the monovalent ions in your assay medium. E.g. for a
typical assay medium with 150 mM NaCl, some 1 mM of chelating
substances, about 1 mM total CaCl2 and 1 mM total MgCl2, plus some 25
mM pH buffers, your final ionic strength would be around 160 ionic
equivalents. So, you should enter this figure in the final ionic
strength box, and modify it when the metal calculations yield medium
ionic strengths differing from 150. Usually, you only need one
iteration to come to a value that is valid for all media you want to
test. An iterative procedure would solve this of course, but
calculation times would increase.
Calculate
The 'Calculate' option of the main menu contains the actual
computing of the program. After you have selected a media
composition, the program first needs to calculate a number of
coefficient arrays and the effects of the experimental conditions on
the stability constants. Therefore, you have to choose 'Prepare
calculations'.
Once the preliminary calculations have been performed, the program
offers the possibilities to calculate total metal cation
concentrations from wanted free concentrations, and the other way
around. The latter procedure is iterative and you may enter a
precision in the 'Special' option of the main menu. Please note that
CHELATOR also gives a medium ionic strength, which has to be equal to
the ionic strength of the monovalent cations in your solutions (see
above).
Special
The 'Special' option of the main menu offers four items. Two of
these ("Complex conc.'s shown" and "Beeping") are toggles (ON/OFF).
'Printer' can also be toggled ON and you will have to specify whether
you are using an Epson-type printer or a HP-type. If you activate
this option and then prepare calculations, a listing of stability
constants and medium conditions will be printed. Otherwise, only
calculation results will be printed. Note: the program tries to
perform a page feed if you toggle the printer option OFF. If the
printer is not connected to the computer at that moment, this may
lead to a 'semi-hangup' of the computer lasting several seconds
(depending on the type and speed of the computer)! "Complex conc.'s
shown" (if ON) results in the display of concentrations of the
metal-chelator complexes. 'Iteration tolerance' allows you to specify
a tolerance for the calculation of free from total metal cation
concentrations. 'Beeping' may be toggled OFF if you are not amused by
a musical PC.
The default datafile, CHELDFLT.DAT
Some peculiarities might need further explanation.
- Beware of metals with many water molecules in their hydration shell
(e.g. Al3+ or Cu2+). CHELATOR offers some ways to account for the
equilibria involved, but these might be inadequate. Look up as many
stability constants as you can and try to reduce some equilibria to
those included in CHELATOR.
- Citrate is listed as having a charge of -3. This represents citrate
with one bound proton. All constants listed refer to binding of ions
to the H+-citrate form. The reason is that citrate binds its first
proton with a K around 16. This first proton will always be bound
under normal physiological conditions. Care should be observed when
multivalent metal ions are to be used: such ions (e.g. Cu2+) might be
able to remove this bound proton, in which case you have to deal with
completely different binding characteristics.
- Amino acids (e.g. aspartate or glutamate) are included in the
default file, but should not be used at high concentrations. Free
metal ion concentrations should be controlled by more specific
chelators. Apart from their non-selectiveness these compounds suffer
from a tendency to form a double complex (MC + C <-> MC2) when
their concentration is high. Since CHELATOR does not account for such
reactions yet, calculations regarding the above conditions may yield
incorrect results.
- Some stability constants of the reaction M + HC <-> MHC (Kd)
are listed as "calculated" in the stability constant table. These had
to be computed from the tabulated stability constants for C + H
<-> HC (Ka), for C + M <-> MC (Kb) and for MC + H
<-> MHC (Kc) by the formula Kd = Kb + Kc - Ka. Note that (as
everywhere else in the program) logarithms of the actual stability
constants are used; this is why the constants are added, not
multiplied.
Last remarks
This should cover most of the operation of CHELATOR. If you have
questions or suggestions, do not hesitate to contact me. An update
with suggested changes will become available to registered users
(please remember: no fees are involved in registering!) in about one
year. Apart from the suggested improvements it will certainly
implement much improved windowing (it will be implemented in Turbo
Pascalþ 6.0 using Turbo Vision).
DISCLAIMER
I seem to have to write the following lines in order to avoid any
unwanted consequences of distributing my little creation.
I will not be liable for any damages, including lost profits, lost
savings, lost time, lost data, wrong experiments, or other incidental
or consequential damages arising out of the use of or inability to
use CHELATOR, even if I have been advised of the possibility of such
damages, of for any claim by any other party.
Th. Schoenmakers
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