Table 2.
Steroid-Binding Affinities and Specificities of Wild-Type SHBG and SHBG Mutants
| SHBG | Kd, nM | Relative Binding Activity (% of DHT) |
|
|---|---|---|---|
| Testosterone | Estradiol | ||
| Wild type | 0.30 ± 0.08 | 24.02 ± 4.18 | 4.52 ± 0.57 |
| R(22)H | 0.33 ± 0.02 | 23.23 ± 5.34 | 4.64 ± 0.87 |
| T7N | 0.30 ± 0.07 | 21.83 ± 1.72 | 4.75 ± 1.65 |
| T48I | 0.78 ± 0.10a | 23.88 ± 4.69 | 5.32 ± 0.58 |
| E52Q | 0.24 ± 0.03 | 21.43 ± 2.04 | 4.50 ± 0.54 |
| R94Q | 0.35 ± 0.01 | 24.80 ± 4.82 | 4.73 ± 0.88 |
| L95M | 0.30 ± 0.02 | 24.50 ± 4.54 | 4.86 ± 0.52 |
| D110N | 0.39 ± 0.19 | 21.14 ± 0.12 | 4.51 ± 0.16 |
| E119D | 0.31 ± 0.04 | 24.81 ± 3.94 | 4.53 ± 0.89 |
| R123C | 0.49 ± 0.05a | 24.48 ± 3.53 | 5.82 ± 0.36b |
| R123H | 1.29 ± 0.23a | 26.26 ± 4.49 | 5.42 ± 0.34c |
| R135C | 0.27 ± 0.01 | 25.93 ± 3.36 | 7.64 ± 0.94a |
| A150P | 0.40 ± 0.17 | 25.58 ± 4.41 | 5.13 ± 0.97 |
| N152K | 0.28 ± 0.06 | 23.91 ± 1.99 | 4.97 ± 0.70 |
| R154W | 0.33 ± 0.03 | 24.38 ± 4.36 | 3.97 ± 0.71 |
| L165M | 0.24 ± 0.04 | 22.93 ± 1.71 | 7.37 ± 0.32a |
| E176K | 0.35 ± 0.04 | 23.59 ± 0.44 | 8.62 ± 0.47a |
| S192L | 0.27 ± 0.07 | 25.79 ± 1.13 | 4.53 ± 0.61 |
| G195E | 1.52 ± 0.08a | 21.72 ± 3.34 | 3.94 ± 0.62 |
Kd values of wild-type SHBG and SHBG mutants for DHT were determined by Scatchard analysis using [3H]DHT as labeled ligand. The relative binding affinity values of SHBG for testosterone and estradiol were determined as percentages by dividing the IC50 of DHT (ie, the concentration of DHT required to displace 50% of [3H]DHT from SHBG) with the corresponding IC50 values of unlabeled testosterone and estradiol in standard competitive steroid-binding assays (27). The Kd and relative binding affinity values are expressed as means ± SD of 3 independent assays.
a
P < .001.
b
P < .01.
c
P < .05.