9
Download
1 / 13

9 th PBL in Calcium and Phospholipid Signaling Department of Clinical Science and Education; - PowerPoint PPT Presentation


  • 70 Views
  • Uploaded on

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

loader
I am the owner, or an agent authorized to act on behalf of the owner, of the copyrighted work described.
capcha
Download Presentation

PowerPoint Slideshow about ' 9 th PBL in Calcium and Phospholipid Signaling Department of Clinical Science and Education;' - gavril


An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -
Presentation Transcript

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


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+


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


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


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


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


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


ad