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9 th PBL in Calcium and Phospholipid Signaling Department of Clinical Science and Education; Department of Physiology and Pharmacology. May 4-15, 2009. Md. Shahidul Islam, M.D., Ph.D. Associate Professor Department of Clinical Research and Education Karolinska Institutet

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slide1

9 th PBL in

Calcium and Phospholipid Signaling

Department of Clinical Science and Education;

Department of Physiology and

Pharmacology. May 4-15, 2009

Md. Shahidul Islam, M.D., Ph.D.

Associate Professor

Department of Clinical Research and Education

Karolinska Institutet

Forskningscentrum, Södersjukhuset

118 83 Stockholm, Sweden

[email protected]

preparation of multiple ligand ion solutions

[L.Ca]

Ka

=

[L].[Ca2+]

Preparation of multiple ligand-ion solutions

Association constant or binding constant

L + Ca2+

L.Ca

dissociation constant

[L].[Ca2+]

Kd

=

[L.Ca]

At half saturation of the ligand: [L]=[L.Ca]

And Kd = [Ca2+]

Dissociation constant

Kd=1/Ka

there are many ca 2 buffers to choose from
There are many Ca2+ buffers to choose from
  • EGTA
  • BAPTA
  • Dibromo BAPTA
  • Quin 2
  • Fura-2
  • EDTA
affinity of the ligand for ca 2 depends on
Affinity of the ligand for Ca2+ depends on
  • pH
    • EGTA more than BAPTA
  • Temperature
  • Ionic strength =0.5SCi.׀Zi׀
how do i know which buffer to use
How do I know which buffer to use?
  • Kd should be close to the desired [Ca2+]
  • Affinity for Ca2+ must be much higher than Mg2+
  • pH-sensitivity of affinity for Ca2+
  • Costs etc
slide8

Ca2+ buffers exist in multiple states of protonation

[Lt]-[CaL]=[L]+[HL]+[H2L]+[H3L]+[H4L]

At pH 6 to just over 7, 99% of EGTA is in the form of H2EGTA

Only two forms of EGTA, EGTA4- and EGTA3- bind Ca2+

slide9

Some important properties of Ca2+ buffers

Ca2+ buffer

Kd

KCa/KMg

EGTA

67 nM

6.2

72.2

KCa(pH 7.4)

(pH 7.4)

BAPTA

192 nM

1.14

158.24

KCa(pH 7.0)

DiBromo

BAPTA

1.83 mM

1.02

63.00

slide10

EGTA is the ”devil we know”

  • Kd near intracellular [Ca2+]i
  • 100,000 times higher affinity for Ca2+ over Mg2+
  • Marked pH-dependence of Ca2+-affinity
  • Variable purity of EGTA
  • 30 times cheaper than BAPTA
  • EGTA itself may have non-specific effetcs
  • on processes studied
slide11

A. Multiple Ligand Approach

When solutions contain more than one ligand

Use computer programs

Two methods of making Ca2+ buffers

B. Two solutions approach

Mix two solutions in various proportions

Single ligand

Ca2+ calibration solutions

slide12

Making of Ca2+ buffers

  • Use High Purity Water
  • Use high purity EGTA, BAPTA
  • Use Plastic Wares
  • Use Ca2+ standard solutions
  • And not CaCl2.2H2O
  • Accuracy in measurement of pH
  • Check by Ca2+ eletrodes
slide13

Lowering Extracellular [Ca2+]

A. Reducing to <1 mM

Stock solution of 1 M Na2H2EGTA

(pH 7.4)

Add Na2H2EGTA three times the concentration

Of Ca2+

B. Reducing Ca2+ to 0

Nominally Ca2+-free medium

Add Na2H2EGTA, 1 mM

C. Precisely known concentration of Ca2+

Use computer programs

Check with Ca2+ electrodes

Or use two solutions approach

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