08-09-2019, 01:05 PM
An extract of Appendix: Calculation of Free Ca, from Use of a Potential Sensitive Dye in Studies on Phospholipid and Sarcoplasmic Reticulum Vesicles, in Enzymes, Receptors and Carriers of Biological Membranes {A Laboratory Manual}, pgs. 142-144, © by Springer-Verlag Berlin Heidelberg 1984.
"This section describes a simple program to calculate the free calcium concentration in solutions buffered with Egta. It is based on a routine developed by M. Pressler, who was so kind to let us use his notes and saved us the work to develop it ourselves. The program yields reliable results as long as the influence of Mg, ATP, and small cations is negligible. It is based on the following equation:
[free Ca] = [CaEgta] / K' (Ca) * [total Egta]
Where K' (Ca) is the apparent stability constant (pH: 7.0) : 10"'6.312
or often given as log K' (Ca) : 6.312
(The value of this constant is currently under dispute. It is advisable to examine the future literature for updated determinations of it)
p (Ca) : -log [free Ca]
The program runs on a HP 65 programmable pocket calculator. It returns the following results:
[free Ca], [CaEgta], and [free Egta] (all in M)
It is started by the following steps:
1) Type in K' (Ca) ==) press A
2) Type in [total Ca] ) press B
3) Type in [total Egta] ) press C
4) Press D to calculate [free Ca]
5) Press E to display [free Egta]
6) Press R/S to display [CaEgta]
Listing of Program Steps
Before typing in the program steps or entering the program stored on a magnetic card initialize the calculator by clearing the registers, the stack and the resident subroutines (f STK, f REG, f PRGM)
BEST!
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"This section describes a simple program to calculate the free calcium concentration in solutions buffered with Egta. It is based on a routine developed by M. Pressler, who was so kind to let us use his notes and saved us the work to develop it ourselves. The program yields reliable results as long as the influence of Mg, ATP, and small cations is negligible. It is based on the following equation:
[free Ca] = [CaEgta] / K' (Ca) * [total Egta]
Where K' (Ca) is the apparent stability constant (pH: 7.0) : 10"'6.312
or often given as log K' (Ca) : 6.312
(The value of this constant is currently under dispute. It is advisable to examine the future literature for updated determinations of it)
p (Ca) : -log [free Ca]
The program runs on a HP 65 programmable pocket calculator. It returns the following results:
[free Ca], [CaEgta], and [free Egta] (all in M)
It is started by the following steps:
1) Type in K' (Ca) ==) press A
2) Type in [total Ca] ) press B
3) Type in [total Egta] ) press C
4) Press D to calculate [free Ca]
5) Press E to display [free Egta]
6) Press R/S to display [CaEgta]
Listing of Program Steps
Before typing in the program steps or entering the program stored on a magnetic card initialize the calculator by clearing the registers, the stack and the resident subroutines (f STK, f REG, f PRGM)
Code:
(-- _:_ -> Enter K' (Ca) (apparent stability constant for CaEgta) using:
LBL 23 10 ENTER log K' (Ca) g y"x; press A)
A 11
DSP 21 (set calculator display format to 3 decimal digits)
3 03
STO 1 33 01 (store K' (Ca) in reg. 1)
RTN 24
(- - - -) Enter [total Ca] in M; press B)
LBL 23
B 12
ST0 2 33 02 (store [total Cal in reg. 2)
RTN 24
(- - - -) Enter [total Egta] in M; press C)
LBL 23
C 13
ST0 3 33 03 (store [total Egta] in reg. 3)
RTN 24
(- - - -) Press D to calculate [free Ca] from:
LBL 23 K' (Ca) = [CaEgta] / [free Cal * [free Egta]
D 14 (solving of the resulting quadratic equation)
RCL 1 34 01
RCL 2 34 02
* 71
RCL 1 34 01
RCL 3 34 03 (Calculate: K' (Ca) * [total Cal + K' (Ca) * [total
* 71 Egta] +1 and store in reg. 4)
+ 61
1 01
+ 61
ST0 4 33 04
f ^-1 32
X^ .5 09 (square above expression and store in reg. 5)
ST0 5 33 05
RCL 1 34 01
f ^ -1 32
x^ .5 09
4 04
* 71 (calculate:
RCL 2 34 02 ((K' (Ca) * [total Ca]+K' (Ca) * [total Egta]+ 1)" 2-
* 71 4K' (Ca) A 2*[total Ca]*[total Egta]) A.S
RCL 3 34 03
* 71
CHS 42
RCL 5 34 04
+ 61
f 31
x^ .5 09
CHS 42
RCL4 34 04
+ 61
2 02 (calculate [CaEgta) and store in reg. 6)
/ 81
RCL 1 34 01
/ 81
ST06 33 06
CHS 42
RCL2 34 02 (calculate and display [free Ca))
+ 61
RTN 24
(-- --) Press E)
LBL 23
E 15
RCL6 34 06 (display [CaEgta])
R/S 84
(-- --) Press R/S)
CHS 42
RCL3 34 03
+ 61 (display [free Egta])
RTN 24
(for next [free Cal in the same solution after adding Ca enter
new total amount of Ca, press B, press D to calculate new
[free Cal.
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