Find Free Mode - Example Calcium calculation

Solution A with a pH 7.4 with ionic equivalent 100 mM at 20°C containing:

• 5 mM EGTA (98% purity)
• 1 mM CaCl2
• 2 mM MgATP (97% purity)
• 2 mM MgCl2

Steps for free Calcium calculation in PyChelator:

1. From "Constants" dropdown, select the preferred constants' source (ex.NIST)
2. Select mM as Input unit.
3. Write the Temperature as 20°C.
4. Write the pH as 7.4.
5. Write the Ionic (Ie) equivalent as 100 mM. If you do not know the ionic equivalent, you can calculate by using the "Calculate Ie" button.
6. Select the parameters to "Show in report", or keep defaults.
7. Using the top panel, write the Chelator concentration of ATP to 2 mM (97% purity), and EGTA to 5 mM (98% purity).
8. Write the Total metals given to the solution; Ca²⁺ as 2 mM and Mg²⁺ as 4 mM
9. Click Calculate. You can copy the Free (M) concentration for Ca²⁺ or the -log10[free]. Alternatively, click Download to have a local copy of the results.

Manual entry of stability constants - Example

Using the stability constants for Zinc and Calcium buffered using EDTA and EGTA, from NIST Critical Stability Constants. A useful table of frequently used chelators and metals can also be found under Maxchelator.

Steps for manual entry of constants in PyChelator:

1. From "Constants" dropdown, select "Write custom constants"
2. Write the Ligands (L1, L2) comma-separated as: EDTA, EGTA.
3. Write the Metals (M1, M2) comma-separated as: Zn, Ca.
4. Write the temperature that the constants were determined (Et) in °C: 20.
5. Write the ionic equivalent that the constants were determined (Ei) in mM: 100.
6. Write the protonation constant H1 (logK) for L1 and L2: 10.3, 9.5
7. Write the ΔH1 enthalpy (kcal/mol) for H1 for L1 and L2: -5.6, -5.9
8. Repeat steps 6 and 7 for all protonation constants.
9. Write the stability constant ML (logK) for M1-L1,M1-L2,M2-L1,M2-L2: 16.6, 12.7, 10.7, 10.9
10. Write the ΔML enthalpy (kcal/mol) for M1-L1,M1-L2,M2-L1,M2-L2: -4.7, -4.3, -6.1, -8.4
11. Repeat Steps 9 and 10 for MHL and ΔMHL, respectively.
12. Download the JSON file. You can use this file the next time using the command Upload constants under Constants dropdown.

Stability constant references

1. NIST: The National Institute of Standards and Technology Database #46 v8. These constants are used in MaxChelator
2. Chelator by Schoenmakers et al. These constants are used in MaxChelator from Ref.
3. SPECS by Fabiato. Taken from Fujushiro et al. 1995 and compared to the reported constants in McGuigan et al.. The ATP ML and MHL constants were corrected for 20°C.
4. Calcium by Föhr et al. 1993