Ligand Proteoglycan Binding

1. The output data will represent the amount of bFGF retained on the filter for a given added concentration. The amount retained in the absence of E-HS should be subtracted from that with E-HS to generate data that represent specific E-HS bound bFGF.

2. Varying the concentrations of bFGF while holding constant the concentration of E-HS (with no inhibitor), this data set should be analyzed to determine the binding capacity (amount of bFGF bound per unit of E-HS) and the binding affinity (KD1), assuming a simple monovalent binding reaction, p + l -kd1- c i which, at steady state assuming negligible ligand depletion, is described by

C1 rtlo kdi + Lo where RT is the binding capacity, and L0 is the concentration of FGF added. The programming to determine KD1 and RT is outlined below.

3. Mathematica software (Version 3.0, Wolfram Research) is launched, and a new notebook page is initialized. Commands as outlined below are entered into the notebook page, and the "shift" and "enter" keys are pressed simultaneously at the end of each instructional set (symbolized in the procedure by " ♦ ")

4. The statistical package must be loaded by typing (see Note 2):

<<StatisticsvNonlinearFif ♦

5. Enter the volume of test solution (in liters) and the molecular weight of the growth factor (bFGF) being investigated:

6. Enter the amount of bFGF added per test well (L0) (ng)

Fadd = {0.05, 0.05, 0.05, 0.05, 0.05, 0.05, 0.10, 0.10, 0.10, 0.10, 0.10, 0.10, 0.20, 0.20, 0.20, 0.20, 0.20, 0.20, 0.20, 0.20, 0.20, 0.20, 0.20, 0.20, 0.50, 0.50, 0.50, 0.50, 0.50, 0.50, 0.50, 0.50, 0.50, 0.75, 0.75, 0.75, 1.0, 1.0, 1.0, 1.0, 1.0, 1.0, 1.0, 1.0, 1.0, 2.0, 2.0, 2.0, 2.0, 2.0, 2.0, 2.0, 2.0, 2.0, 2.5, 2.5, 2.5, 4.0, 4.0, 4.0, 5.0, 5.0, 5.0, 5.0, 5.0, 5.0, 6.0, 6.0, 6.0, 10.0, 10.0}; ♦

7. Enter the measured values of bFGF retained (C1) (ng)

Fbound = {.0025, .0035, .0034, .0013, .0055, .0033, .0067, .0069, .0076, .0028, .0033, .0035, .017, .020, .018, .0063, .0076, .0063, .017, .016, .018, .016, .017, .017, .079, .034, .043, .029, .034, .032, .021, .022, .020, .047, .056, .057, .080, .13, .099, .13, .071, .078, .037, .039, .086, .088, .066, .070, .21, .24, .19, .17, .16, .16, .25, .20, .22, .32, .32, .30, .17, .37, .27, .22, .20, .17,

8. Convert these values (Lo and C1) from ng to nM and store as data sets 1 and 2:

dat2 = Fbound/(volume*mwF);

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