Table 2.
Gel exclusion chromatography of tubulin
| Tubulin conc. (nM), incubation (h) | Apparent MW dimer peak (kDa) | Apparent MW monomer peak (kDa) | % Monomer observed | % Monomer calculated for Kd 1 × 10−11 M |
|---|---|---|---|---|
| 2.0 (ASAP) | 110–116 | 0 | 3.4a | |
| 2.0 (5) | 104–118 | 69–72 | 7–10 | 6.8 |
| 0.2 (ASAP) | 103 | 61 | 7 | 3.7a |
| 0.2 (1.5, 3, 6) | 109 | 70 | 13 | 20 |
| 0.2 (3) | 109 | 69 | 5 | 20 |
| 0.2 (3) | 108 | 71 | 10 | 20 |
| 0.04 (3) | 109 | 73 | 32 | 39 |
| 0.04 (3) | 105 | 47 | ca, 60 | 39 |
| 0.04 (2) | 109 | 51 | ca, 40 | 39 |
| 0.02 (3) | 125 | 82 | 54 | 50 |
| 0.02 (3) | 120 | 79 | ca, 40 | 50 |
| 0.02 (3) | 100 | 69 | ca, 50 | 50 |
a
It was assumed that 10 min elapsed between the time when the tubulin was diluted and when chromatographic separation of the monomer and dimer started; it required about 5 min to filter the protein and start the chromatography. The monomer concentration at 10 min was calculated from Eq. 3, assuming dissociation and association rate constants equal to 6.3 × 10−5 s−1 and 6.3 × 106 M−1 s−1, respectively.