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| 1 | +Titraion of carboxybenzenesulfonamide (CBS) into carbonic anhydrase II (CAII) |
| 2 | + |
| 3 | +06 May 2011 |
| 4 | + |
| 5 | +These experiments follow the control experiment performed in Myszka et al. ABRF MIRG'02. |
| 6 | + |
| 7 | +PREPARATION PROTOCOL |
| 8 | + |
| 9 | +* CAII |
| 10 | + |
| 11 | +Carbonic anhydrase II was obtained from Sigma-Aldrich (product C2522-25mg, lot #XXXXXXXXXXXX) in the form of lyophilized crystals. |
| 12 | +4 mL of PBS (pH 7.4) buffer were dispensed from a 5 ml serological pipette into the container, gently pipetting to dissolve the protein. |
| 13 | +1 mL was transferred into a 2.5 ml dialysis tube, to which an additional 1 mL of PBS buffer was added. |
| 14 | +This tube was dialyzed overnight in a covered beaker filled with ~1 L PBS dialysis buffer. |
| 15 | +Volume levels in the dialysis tube were visually inspected after dialysis and found to be similar. |
| 16 | + |
| 17 | +To roughly assess the protein concentration post-dialysis, a 1:8 dilution was prepared by adding 20 uL of protein solution to 140 ul of PBS dialysis buffer and loading 100 uL into a cuvette with 10 mm path length and assayed in a UV-vis at 280 nm. |
| 18 | +The absorbance was read to be 5.209 +- 0.1 A on the NanoDrop ND-1000. |
| 19 | +Using an extinction coefficient of 50070 M^{-1} cm^{-1} provided by Sigma-Aldrich, this yielded a concentration of ~ 100 uM. |
| 20 | + |
| 21 | +The 2 mL dialyzed protein solution was extracted from the tube and diluted with 8 mL dialysis PBS buffer to yield 10 mL of ~ 10 uM stock. |
| 22 | +The concentration of this ~ 10 uM stock was assayed measuring the absorbance at 280 nm on the NanoDrop ND-1000, which was measured to be 0.457 +- 0.009 A, yielding a concentration of ~9.1 uM. |
| 23 | + |
| 24 | +The dialysis buffer was recovered and filtered and stored at 4 C for use in preparing the CBS solution and in blank titrations. |
| 25 | +pH re-measurement at ambient temperature verified the pH remained at 7.4. |
| 26 | + |
| 27 | +* CBS |
| 28 | + |
| 29 | +Carboxybenzenesulfonamide (CBS) was obtained from Sigma-Aldrich (4-sulfonylbenzoic acid at 97% purity, product 000944798, lot #MKBF3323V). |
| 30 | +To prepare a ~ 2 mM stock solution, 24.8 +- 0.1 mg were massed out onto a tared plastic weighing boat on an analytical balance. |
| 31 | +The contents of the boat were added to a 50 ml Falcon tube, to which 34 +- 0.5 ml dialysis PBS buffer was added using a 50 ml serological pipette. |
| 32 | +And additional 249 +- 2 ul PBS buffer was added using a 1000 ul micropipette, rinsing the boat to ensure all CBS was transferred into the Falcon tube. |
| 33 | +The boat was again rinsed with 1000 ul of the contents of the Falcon tube using the same micropipette. |
| 34 | +The Falcon tube was vortexed vigorously for ~ 10 min to ensure all visible solid was dissolved. |
| 35 | + |
| 36 | +For this ~ 2 mM stock, a total of 24.8 +- 0.1 mg was dissolved in 34.2 +- 0.5 mL PBS, for a total concentration of 0.403 +- 0.007 g/L (~1.7% accuracy). |
| 37 | +Using a MW for CBS of 201.2 g/mol, this gave a solution of 2.00 +- 0.03 mM. |
| 38 | + |
| 39 | +The concentration of the ~ 2 mM stock was assayed using a NanoDrop ND-1000 spectrophotometer to determine the absorbance at 272 nm. |
| 40 | +A ~ 5 ul sample was loaded, and the absorbance was determined to be 0.260 +- 0.005 A using the path length of 1 mm. |
| 41 | +Using an extinction coefficient of 1307 +- 126 M^{-1} cm^{-1} from Myzska et al., the concentration is determined to be 2.0 +- 0.2 mM. |
| 42 | + |
| 43 | +To prepare ~446 uM CBS for loading in the syringe, 0.892 +- 2 ul of 2 mM CBS stock solution was dispensed into a 10 ml Falcon tube using a 200 ul pipetteman, to which 3.109 +- 5 uL ml dialysis PBS buffer was added using a 5000 ul pipetteman. |
| 44 | +The resulting solution should have a concentration of 446 +- 1 uM by propagation of error. |
| 45 | + |
| 46 | +Concentration of the ~ 446 uM for loading in the syringe was assayed using a NanoDrop ND-1000 spectrophotometer to determine the absorbance at 272 nm. |
| 47 | +A ~ 5 ul sample was loaded, and the absorbance determined to be 0.062 +- 0.001 A using the path length of 1 mm. |
| 48 | +Using an extinction coefficient of 1307 +- 126 M^{-1} cm^{-1} from Myzska et al., the concentration is determined to be 474 +- 46 uM. |
| 49 | + |
| 50 | +The concentration was also checked using a more accurate UV-vis spectrophotometer (model???) using a cuvette with a 10 mm path length. |
| 51 | +The absorption at 272 nm was measured to be 0.59667 +- ?????? A, giving a concentration of 457 +- ??? uM. |
| 52 | +[JDC: Check this.] |
| 53 | + |
| 54 | +ITC PROTOCOL |
| 55 | + |
| 56 | +Syringe: [CBS] 443 uM |
| 57 | +Sample cell: [CAII] 9.13 uM |
| 58 | + |
| 59 | +10 injections of 10 uL were used, with a 10 uL "syringe down" command issued prior to loading syringe into VP-ITC. |
| 60 | +A 300 s pre-injection data recording period was used |
| 61 | +Each injection was 20 s in duration, with 240 s between injections. |
| 62 | +The experiment was performed at 25 C, with samples equilibrated to ~ 24 C prior to loading. |
| 63 | +The room in which the instrument is located was thermostatted to 21 C. |
| 64 | + |
| 65 | +CBS concentration determined from absorbance at 272 with NanoDrop ND-1000 spectrophotometer, using extinction coefficient 1307 +- 126 M^{-1} cm^{-1} from Myszka paper. |
| 66 | +CAII concentration determined from absorbance at 280 nm with NanoDrop ND-1000 spectrophotometer, using extinction coefficient 50070 M^{-1} cm^{-1}. |
| 67 | +Both CBS and CAII were dissolved in PBS pH 7.4 buffer from overnight dialysis of CAII sample. |
| 68 | + |
| 69 | +MANIFEST |
| 70 | + |
| 71 | +original-data/ - original .itc files produced by the MicroCal VP-ITC |
| 72 | +origin-power-measurements/ - text file of Origin extracted power measurements (filter time 2 s) |
| 73 | +origin-baseline-fits/ - PDFs of Origin automated baseline fits |
| 74 | +origin-integrated-heats/ - text files of Origin automatic integration of heats |
| 75 | +origin-fit/ - PDF of Origin automated fit of titration of ligand into protein (without blank subtraction) |
| 76 | + |
| 77 | +EXPERIMENTS |
| 78 | + |
| 79 | +050611a - titration of CBS into CAII |
| 80 | +050611b - titration of buffer into CAII |
| 81 | +050611c - titration of buffer into buffer |
| 82 | +050611d - titration of CBS into buffer |
| 83 | + |
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