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Which of the following amino acids would be first to elute at pH 7 from an anion-exchange column?...
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Transcript of Which of the following amino acids would be first to elute at pH 7 from an anion-exchange column?...
![Page 1: Which of the following amino acids would be first to elute at pH 7 from an anion-exchange column? a). glutamic acid b). alanine c). lysine d). asparagine.](https://reader036.fdocuments.us/reader036/viewer/2022082816/56649d825503460f94a672ae/html5/thumbnails/1.jpg)
Which of the following amino acids would be first to elute at pH 7 from an anion-exchange column?a). glutamic acidb). alaninec). lysined). asparaginee). glycine
![Page 2: Which of the following amino acids would be first to elute at pH 7 from an anion-exchange column? a). glutamic acid b). alanine c). lysine d). asparagine.](https://reader036.fdocuments.us/reader036/viewer/2022082816/56649d825503460f94a672ae/html5/thumbnails/2.jpg)
A mixture of lysine, glycine, alanine, isoleucine and glutamic acid are separated by ionic exchange chromatography. What is the order of elution of these amino acids if :
Use a cation exchange resin?
b) use an anion exchange resin?
Which column would give the best separation?
![Page 3: Which of the following amino acids would be first to elute at pH 7 from an anion-exchange column? a). glutamic acid b). alanine c). lysine d). asparagine.](https://reader036.fdocuments.us/reader036/viewer/2022082816/56649d825503460f94a672ae/html5/thumbnails/3.jpg)
Glycine, alanine, valine and leucine can be successfully separated by ionic exchangechromatography even though their pKas are almost identical. Explain the behavior of these amino acids.
![Page 4: Which of the following amino acids would be first to elute at pH 7 from an anion-exchange column? a). glutamic acid b). alanine c). lysine d). asparagine.](https://reader036.fdocuments.us/reader036/viewer/2022082816/56649d825503460f94a672ae/html5/thumbnails/4.jpg)
Since these four amino acids can be separated by ion exchange chromatography, but that their pI are virtually identical, another property must be responsible for this behavior.
A close look at the structure of glycine, alanine, valine and leucine reveals a progressive increase in the hydrophobic character of their side chain. We can deduce that these amino acids can establish hydrophobic interactions with the ion exchange resin, allowing their separation.
![Page 5: Which of the following amino acids would be first to elute at pH 7 from an anion-exchange column? a). glutamic acid b). alanine c). lysine d). asparagine.](https://reader036.fdocuments.us/reader036/viewer/2022082816/56649d825503460f94a672ae/html5/thumbnails/5.jpg)
Consider the following peptide: A-L-K-M-P-E-Y-I-S-T-D-Q-S-N-W-H-H-R Indicate the fragments generated after the following digestions : a) trypsin
b) chymotrypsin
![Page 6: Which of the following amino acids would be first to elute at pH 7 from an anion-exchange column? a). glutamic acid b). alanine c). lysine d). asparagine.](https://reader036.fdocuments.us/reader036/viewer/2022082816/56649d825503460f94a672ae/html5/thumbnails/6.jpg)
Trypsin hydrolyzes the peptide bond on the carboxyl-side of the basic amino acids lysine and arginine. A-L-K M-P-E-Y-I-S-T-D-Q-S-N-W-H-H-R
Chymotrypsin hydrolyzes the peptide bond on the carboxyl-side of the large hydrophobic amino acids F, Y, W, and MA-L-K-M P-E-Y I-S-T-D-Q-S-N-W H-H-R
![Page 7: Which of the following amino acids would be first to elute at pH 7 from an anion-exchange column? a). glutamic acid b). alanine c). lysine d). asparagine.](https://reader036.fdocuments.us/reader036/viewer/2022082816/56649d825503460f94a672ae/html5/thumbnails/7.jpg)
We load a DEAE-cellulose column adjusted to a pH of 6.5 with the following mixture of proteins: ovalbumin (pI = 4.6), urease (pI = 5.0)myoglobin (pI = 7.0).
The proteins are eluted first with a buffer of weak ionic strength at a pH of 6.5, and then the same buffer containing increasing amounts of sodium chloride is used to elute the proteins. What order are the proteins eluted?
![Page 8: Which of the following amino acids would be first to elute at pH 7 from an anion-exchange column? a). glutamic acid b). alanine c). lysine d). asparagine.](https://reader036.fdocuments.us/reader036/viewer/2022082816/56649d825503460f94a672ae/html5/thumbnails/8.jpg)
myoglobin will elute first, since it will not bind to the resin (pH<pI: positive net charge);
the pI of urease (5,4) is closer to the pH of the buffer that is the pI of ovalbumin (4,6): urease will therefore carry a lower number of negative charges than ovalbumin.
We can therefore predict that urease will elute at a NaCl concentration which will be lower than the one required to elute ovalbumin.
![Page 9: Which of the following amino acids would be first to elute at pH 7 from an anion-exchange column? a). glutamic acid b). alanine c). lysine d). asparagine.](https://reader036.fdocuments.us/reader036/viewer/2022082816/56649d825503460f94a672ae/html5/thumbnails/9.jpg)
DEAE cellulose columns are rarely used at pH greater than 8.5. Why?
![Page 10: Which of the following amino acids would be first to elute at pH 7 from an anion-exchange column? a). glutamic acid b). alanine c). lysine d). asparagine.](https://reader036.fdocuments.us/reader036/viewer/2022082816/56649d825503460f94a672ae/html5/thumbnails/10.jpg)
6-phosphogluconate dehydrogenase has a pI of 6. Explain why the buffer used for a chromatography on DEAE-cellulose must have a pH greater than 6 but less than 9 in order to ensure the enzyme is efficiently bound to the column.
![Page 11: Which of the following amino acids would be first to elute at pH 7 from an anion-exchange column? a). glutamic acid b). alanine c). lysine d). asparagine.](https://reader036.fdocuments.us/reader036/viewer/2022082816/56649d825503460f94a672ae/html5/thumbnails/11.jpg)
At a pI above 6, 6-phosphogluconate dehydrogenase has a net negative charge (pH>pI): it will bind the resin.
At a pH value above 9, the diethylamino group of the resin is deprotonated, preventing any separation of the enzyme as a function of its charge.