Read CA6 activity, and review the protein MW ladder Use the data obtained below,
ID: 190546 • Letter: R
Question
Read CA6 activity, and review the protein MW ladder Use the data obtained below, to determine the structural nature of this protein For each experiment, give your analysis, to justify your answer reliminary MW determination - Gel filtration Method tsinga gel tiltration column of Su planne on, ino Mill·PRxp1170. monitor 1 Results will tihtain .t single peak that elutes half was hetween mysin and ,galactosidase GGel clectrophoresis Method tsingpoli acrylamide gel electorloresimo! Mglycine-P1140 Results are not interpretable - Subunit makeup via Gel Filtration Alethd Using a gel tiltration column of Sephatose ot, in u.IM IHILPIS, pl 70 with o M urca tounum hydrochloride), n'ontoringvaV280 Results )ou observe a single peak that elutes about 2%lower than ov albumin - Subunit makeup via SDS PAGE Method Ising poli acrylannde gel electnphoresilno311 Tri.pl ihs, witho5.sls Results you observe a single band that migrates to pont abeut7faster than oalbumn Method Gel tiltration of the isolated 10.000mw subunits on a column of Sephadex G-75, in O.IM HEPIS. pli 7o "ith o M urea tor guanidinium hvdrchlondel and 10mM 2-mercaptoethanol, monitor ng via1280 - Disultide bonds via Gel Filtration You observe two peaks, one that coclutes with carbonic anhydrase and one that elutes ater Iywzyme ts Method Polvacrtlannde gel electrophoresis ot the iolated loooo Mwsubunts in ussilii llos with os SDs tollowing treatment of the subunits wath 10mM 2-mercaptoethanol Results you observe two bands, one that Disultide bonds via SDS-PAGE& 2ME nd one that murates taster than IvsozymeExplanation / Answer
Answer = Total MW 41875Da and contains 2 subunits, one with MW - 31000Da and other with 10875Da. Both are joined by disulfide bond.
Total MW identified from EXP1 preliminary MW determination.
Single peak half way between myosin and beta-gal
So, (200000 - 116250 ) / 2 = 41875 Da.
Toatal MW Confirmation by 2 experiments,
Subunit makeup via gel filtration in this single peak that elutes 7% slower than ovalbumin. That means Ovalbumin 45000 Da. 7% means= 45000 X 0.07 = 3150
45000 - 3150 = 41850 Da.
In gel filtration large molecules moves fast than small molecues. Thats why 41850Da molecules elutes 7% slower than 45000 Da molecule.
Another method to confirm Total MW - In subunit makeup Via SDS page.
Small molecule move faster in SDS page than larger molecule. Thats why here 41850Da molecules elutes 7% faster than 45000 Da molecule.
To find how many subunits present in the structure by DIsulfide bons via Gel filtartion
Here mercaptoetanol breaks disulphide bond and elutes 2 bands, one with carbonic anhydrase at 31000 Da and other elutes after lysozyme. It clearly shows other molecule less than lysozyme MW 14400.
41875-31000 = 10875 MW for other molecule.
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