estimate the molecular weight of sphingosine kinase The investigators carried ou
ID: 477016 • Letter: E
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estimate the molecular weight of sphingosine kinase The investigators carried out several additional chromatographic steps and finally obtained a fraction that contained pure sphingossine kinase, as determined by SDS- PAGE. The molecular weight was determined by comparing the migration of the enzyme with the migration of standard molecular weight markers in Lane 1. The investigators also carried out an additional experiment in which one batch of the enzyme was treated with the reducing agent beta-mercaptoethanol (Lane 2). A second batch was not treated (Lane 3). The results are shown in Figure 19.5. Estimate the molecular weight of sphingosine kinase. Interpret the results of the experiment described above What can you say about the structure of sphingosine kinase?Explanation / Answer
a. The molecular weight of sphingosine kinase is approximately 48 Kd.
b. One batch of sphingosine kinase was treated with the reducing agent beta-mercaptoethanol, which breaks the disulphide bonds that may form between thiol groups of cysteine residues
$$RS-SR + 2HOCH_2CH_2SH ightleftharpoons HOCH_2CH_2S-SCH_2CH_2OH+2RSH$$
When S-S bonds are broken, both tertiary and quaternary structures can be affected. Thus dimers or oligomers linked through S-S bond may break in monomers. In the above experiments, the molecular weight of the enzyme sphingosine kinase in lanes 2 and 3 is same, this ensures that the enzyme sphingosine kinase contains only monomers. No dimers or oligomers are present.
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