8.5 Radioligand binding studies
8.5.3 Saturation experiments
([³H]isoBRV), or four individual experiments performed in duplicate ([³H]BRV), respectively.
Table 16: Conditions for kinetic binding studies.
[³H]LEV [³H]isoBRV [³H]BRV
buffer solution Tris-HCl buffer containing MgCl2 (2 mM)
radioligand concentration 10 nM 5 nM 1 nM
amount of protein per well 200 µg 100 µg 100 µg
incubation time until dissociation was started
120 min 180 min 240 min
− = %&'( ∙
#$+
Equation 7: Mathematical function of saturation binding curve
R-L: receptor-ligand complex ≙ Bound
Bmax: maximum number of binding sites (cpm) L: unbound ligand ≙ Free
KD: equilibrium dissociation constant (M)
As indicated in Figure 43 the KD value corresponds to the concentration of radioligand at which 50% of the receptors are occupied, whereas Bmax equals the value that is asymptotically approximated by the hyperbola. Bmax (cpm) can subsequently be used for the calculation of Bmax (pmol/mg protein) (Equation 8 to Equation 10):
)* %+,-. = %&'( ( )0)
2 (0 ) ∙ 3445 53- 6 (%) ∙ 2.2 ∙ 9)3 . : 5;5 6 (<5/00+ )
Equation 8: Calculation of pM Bound
Bmax: maximum number of binding sites (cpm) V: volume per well (ml)
efficiency: counter efficiency (cpm/dpm ≙ cpm in %) 2.2: factor for converting cpm in Bq
for details see Deupree et al.155
spec. activity: specific activity of radioligand (Ci/mmol)
%&'( ()0+ /=3 ) = )* %+,-. ∙ 2 ( )
Equation 9: Calculation of Bmax (pmol/well)
%&'( ()0+ /0> )?+ 35-) = %&'( ()0+ /=3 ) )?+ 35- (0>/=3 )
Equation 10: Calculation of Bmax (pmol/mg protein)
By plotting the concentration of bound radioligand against the quotient of bound/free radioligand (see Figure 43 B) a Rosenthal plot is obtained.233 In case of ligand binding to a single site a linear correlation can be observed. Thereby, the slope of the line equals -1/KD, while the x-intercept corresponds to the value of Bmax. If the ligand is binding to
more than one binding site, the Rosenthal plot will deviate from a linear function and express a bent curve. However, it should be mentioned that the Rosenthal plot should only be used for visualizing not analyzing data, since the transformation is more prone to errors.
For saturation experiments, in which intact cells instead of membrane preparations are used, Bmax (binding sites/cell) can be calculated using the following equations (Equation 8, Equation 11 and Equation 12):
%&'( ()0+ / 3 ) = )* %+,-. ∙ 2 ( ) 3 9 )3? =3
Equation 11: Calculation of Bmax (pmol/cell)
%&'( (@5-.5-> 95 39/ 3 ) = %&'( ()0+ / 3 ) ∙ 10C ∙ DE
Equation 12: Calculation of Bmax (binding sites/cell)
NA: Avogadro constant (6.022 · 1023)
8.5.3.2 Performance of saturation experiments
All saturation experiments were performed in analogy to the procedure described by Noyer et al.60 The protein preparation was incubated for a certain amount of time at 4 °C in a total volume of 0.5 ml Tris-HCl buffer, containing MgCl2 (2 mM) and the radioligand. Non-specific binding was determined for all radioligand concentrations separately in the presence of unlabeled levetiracetam (1 mM). Separation of bound from unbound radioligand was achieved by filtration through GF/C glass fiber filters pre-soaked for 30 min in 0.1% aqueous PEI solution. Subsequently, it was washed three times with ice-cold Tris-HCl buffer.
8.5.3.2.1 Saturation studies with [³H]LEV at rat brain cortical membrane preparations
The saturation experiment of [³H]LEV at rat brain cortical membrane preparations (RC) was performed with radioligand diluted with a constant percentage of unlabeled levetiracetam (isotopic dilution). This is a convenient method to decrease the amount of radioligand applied in the assay, if unlabeled ligand for dilution is available. In this case
the radioligand [³H]LEV (98 Ci/mmol) was diluted by a factor of 100 according to Equation 13:
?:.5+ 5>:-. (μ<5) = 9)3 . : 5;5 6G (<5/00+ ) ∙ G (-*) ∙ 4 ∙ 2H I I'J (0 ) 1000
Equation 13: Calculation of the amount of radioligand (µCi)
spec. activityd: desired specific activity after dilution (Ci/mmol) Ld: highest desired concentration of radioligand in the assay (nM)
f: assay dilution factor (here: 5, since 100 µl are given into a total volume of 500 µl) VL total: required volume of the highest concentrated radioligand solution
[LM] 2 (μ<5) = 0.98 <5/00+ ∙ 30000 -* ∙ 5 ∙ 1.1 0
1000 = 161.7 μ<5
Since the original radioligand [³H]LEV solution possesses a concentration of 1 mCi/ml according to Equation 13 a total amount of 162 µl of [³H]LEV was needed.
The required amount of unlabeled levetiracetam (mg) for dilution can be calculated by Equation 14:
,- :@3 3. 5>:-. (0>) = * ∙ (1 :G − 1
: )
Equation 14: Calculation of the amount of unlabeled ligand (mg)
M: molecular weight of unlabeled ligand (mg/mmol) L: required amount of radioligand as calculated (mCi) ad: desired specific activity after dilution (mCi/mmol)
ao: specific activity of original radioligand solution (mCi/mmol) 2 (0>) = 170.11 0>/00+ ∙ 0.1617 0<5 ( 1
980 − 1
98000) (00+ /0<5)
= 28 μ>
Consequently, for the preparation of 1100 µl of the highest concentrated radioligand solution 162 µl [³H]LEV and 28 µl of levetiracetam solution (1 mg/ml) were given into 910 µl Tris-HCl buffer.
The series of dilution starting from the highest concentrated radioligand solution was prepared according to the following table:
Table 17: Preparation of dilution series for [³H]LEV solutions (30000-5 nM); f.c.: final concentration in the assay, f: dilution factor with regard to previous solution. From the radioligand solution with the highest concentration (first row), 300 µl are taken (see last column) to prepare the next dilution (second row). Since this requires to dilute the first solution by the factor of 3 (second column), 600 µl of Tris-HCl buffer need to be added (third column). This adds up to a volume of 900 µl (fourth column), from which 130 µl are taken (last column) for the preparation of the next dilution (third row), and so on.
f.c.
(nM) f dilution step
(previous solution + Tris-HCl) (µl)
prepared V (µl)
remaining V (µl)
30000 1100 (-300) = 800
10000 3 300 + 600 900 (-130) = 770
1000 10 130 + 1170 1300 (-550) = 750
500 2 550 + 550 1100 (-260) = 840
100 5 260 + 1040 1300 (-500) = 800
50 2 500 + 500 1000 (-240) = 760
10 5 240 + 960 1200 (-400) = 800
5 2 400 + 400 800
For saturation experiments with [³H]LEV at rat cortical membrane preparations (RC) 200 µg of protein membrane preparation (see 8.3.1) per well were used. The assay was incubated for 120 min. The actual concentration of radioligand in the assay, which was determined by measuring aliquots of each radioligand dilution, was used for plotting the saturation curve. Obtained KD and Bmax values are means of two individual experiments performed in triplicate.
8.5.3.2.2 Saturation studies with [³H]isoBRV at rat brain cortical membrane preparations
Since no unlabeled ligand was available for dilution of the radioligand the saturation experiment with [³H]isoBRV was performed without isotopic dilution. In order to reduce the amount of applied radioligand, the saturation experiment was not performed until a highest concentration of 10 times the expected KD value. Instead, a total amount of 1000 µl of the original radioligand solution in ethanol was vaporized to dryness at ambient pressure over several days. The residue was dissolved in the determined volume of Tris-HCl buffer required for the highest concentration (see Table 18) and the series of dilution was prepared as described in the following table:
Table 18: Preparation of dilution series for [³H]isoBRV solutions; relative f.c.: final concentration in the assay in relation to lowest concentrated solution (second last row), f: dilution factor with regard to previous solution. For a detailed explanation see Table 17.
relative
f.c. f dilution step
(previous solution + Tris-HCl) (µl)
prepared V (µl)
remaining V (µl)
200 1235 (-785) = 450
100 2 785 + 785 1570 (-1116) = 454
80 5/4 1116 + 279 1395 (-945) = 450
60 4/3 945 + 315 1260 (-810) = 450
40 3/2 810 + 405 1215 (-765) = 450
30 4/3 765 + 255 1020 (-570) = 450
20 3/2 570 + 285 855 (-405) = 450
10 2 405 + 405 810 (-360) = 450
5 2 360 + 360 720 (-270) = 450
2 5/2 270 + 405 675 (-225) = 450
1 2 225 + 225 450
0 only Tris-HCl
For saturation experiments with [³H]isoBRV at rat cortical membrane preparations (RC) 100 µg of protein membrane preparation (see 8.3.1) per well were used. The assay was incubated for 180 min. Final concentrations (nM) of the radioligand in the assay were determined by measuring an aliquot of each dilution. The results from two individual experiments performed in duplicate were plotted against determined actual concentrations.
8.5.3.2.3 Saturation studies with [³H]BRV at rat brain cortical membrane preparations
As already described for the saturation experiment with [³H]isoBRV, likewise no isotopic dilution was done for the saturation experiment with [³H]BRV, since no unlabeled ligand was available. For a required radioligand concentration of 650 nM (final concentration in the assay) the amount of original radioligand solution was determined according to Equation 13:
[LM]% 2 (μ<5) = 94 <5/00+ ∙ 650 -* ∙ 5 ∙ 1.37 0
1000 = 419 μ<5
The corresponding amount (419 µl) of original [³H]BRV solution was given into 951 µl Tris-HCl buffer to add up to the determined total volume (1370 µl) for the highest concentration (see Table 19), which was diluted as described in the following table:
Table 19: Preparation of dilution series for [³H]BRV solutions (650-1 nM); f.c.: final concentration in the assay, f: dilution factor with regard to previous solution. For a detailed explanation see Table 17.
f.c.
(nM) f dilution step
(previous solution + Tris-HCl) (µl)
prepared V (µl)
remaining V (µl)
650 1370 (-920) = 450
400 13/8 920 + 575 1495 (-1035) = 460
300 4/3 1035 + 345 1380 (-930) = 450
200 3/2 930 + 465 1395 (-945) = 450
150 4/3 945 + 315 1260 (-810) = 450
100 3/2 810 + 405 1215 (-765) = 450
75 4/3 765 + 255 1020 (-560) = 460
50 3/2 560 + 280 840 (-370) = 470
25 2 370 + 370 740 (-288) = 452
10 5/2 288 + 432 720 (-270) = 450
5 2 270 + 270 540 (-90) = 450
1 5 90 + 360 450
For saturation experiments with [³H]BRV at rat cortical membrane preparations (RC), 100 µg of protein membrane preparation (see 8.3.1) per well were used. The assay was incubated for 240 min. The results from two individual experiments performed in duplicate were plotted against determined actual concentrations.
8.5.3.2.4 Saturation studies with [³H]BRV on intact cells
Saturation experiments with [³H]BRV on intact CHO cells recombinantly expressing the protein of interest were performed as following: transiently transfected CHO cells (see 8.7.6.1 b) were prepared for binding studies as described in 8.7.7. For saturation experiments on CHO cells recombinantly expressing hSV2A-GFP cells of two dishes (152 cm2) grown to confluence suspended in Tris-HCl buffer were used for a 24-well assay, whereas for experiments on CHO cells recombinantly expressing rSV2A-GFP and rSV2A_N364K-GFP the amount of cells was reduced to one confluent dish per
24-well assay. The incubation time for the assay was 240 min. KD and Bmax values are means of two individual experiments performed in duplicate. The radioligand was used without isotopic dilution by vaporizing a certain amount (~500 - 750 µl) of original radioligand solution at ambient pressure over several days and dissolving the residue in a defined volume of Tris-HCl buffer as described in Table 20. Final concentrations (nM) of the radioligand in the assay were determined by measuring an aliquot of each dilution.
Table 20: Preparation of dilution series for [³H]BRV solutions; relative f.c.: final concentration in the assay in relation to lowest concentrated solution (last row), f: dilution factor with regard to previous solution. For a detailed explanation see Table 17.
relative
f.c. f dilution step
(previous solution + Tris-HCl) (µl)
prepared V (µl)
remaining V (µl)
65 912 (-462) = 450
30 13/6 462 + 539 1001 (-540) = 461
20 3/2 540 + 270 810 (-360) = 450
10 2 360 + 360 720 (-270) = 450
5 2 270 + 270 540 (-90) = 450
1 5 90 + 360 450