Fresh fruiting bodies of Stropharia rugosoannulata were homogenized and added to
ID: 277605 • Letter: F
Question
Fresh fruiting bodies of Stropharia rugosoannulata were homogenized and added to 80% (NH4)2SO4. The precipitate was collected by centrifugation, dissolved in and dialyzed against distilled water before applying on a CM-cellulose column pre-equilibrated with 10 mM phosphate buffer (pH 5.2). After removal of the unadsorbed fraction (CM1), the adsorbed fractions (CM2 and CM3) were eluted with 0.15 M and 1 M NaCl in phosphate buffer . Hemagglutinating activity was found to be enriched in fraction CM2. Fraction CM2 was then subjected to a column of Q-Sepharose in 10 mM sodium acetate buffer (pH 6.1). Three fractions (Q1, Q2 and Q3) were collected after elution with 0.05 M, 0.15 M and 1 M NaCl in 10 mM sodium acetate buffer (pH 6.1), respectively.The hemagglutinating activity was enriched in the Q2 . Q2 was then applied on an SP-Sepharose column in 10 mM sodium acetate buffer (pH 5.0). Unbound material was eluted with the starting buffer then bound material was desorbed by addition of 50 mM NaCl, and 1 M NaCl to the starting buffer, respectively . The peak containing hemagglutinating activity was subsequently subjected to gel filtration by fast protein liquid chromatography on a Superdex 75 HR 10/30 column in 0.15 M ammonium bicarbonate buffer (pH 8.5) using an AKTA Purifier.
In the above protocol, precipitation of the carbohydrate binding protein was achieved with a high concentration of ammonium sulphate (80% (v/v)).
Questions
1a. Give a full molecular description rationalising why the protein requires a relatively high concentration of ammonium sulphate for it to precipitate.
1b. The first three chromatography procedures in the purification of the protein employ CMcellulose, Q-Sepharose and SP-Sepharose matrices at pH values 5.2, 6.1 and 5.0, respectively. Name the functional group associated with each of the three matrices, as well as the charge present under the operating conditions
1c. which matrices do you think will be effective in adsorbing the protein with the buffers utilized. Justify your answer.
Q1d. Suggest a pI range likely for the protein. Justify your answer (show your working) with respect to the different buffer and matrix chemistries utilized.
Q1e.Suggest a pI range likely for proteins eluting in fraction Q3. Justify your answer (show your working) with respect to the buffer and matrix chemistries utilized.
Explanation / Answer
1a. Give a full molecular description rationalising why the protein requires a relatively high concentration of ammonium sulphate for it to precipitate.
Ans: Protiens with higher molecular weight and higher hydrophilicity (having greater percentage of hydrophilic amino acids in its peptide) requires higher concentration of salts to precipitate. For a protein to be soluble in aqueous solution there are three kinds of water interaction. ion hydration between charged side chains (e.g., Asp, Glu, Lys), hydrogen bonding between polar groups and water (e.g., Ser, Thr, Tyr, and the main chain of all residues), and hydrophobic hydration (Val, Ile, Leu, Phe). With the percentage increase in polar aminoacids like Ser, Thr, Tyr in a protein it takes more salt to precipitate out. If the percentage of hydrophobic amino acids is more in a given protien it takes less salt to precipitate.
1b. The first three chromatography procedures in the purification of the protein employ CMcellulose, Q-Sepharose and SP-Sepharose matrices at pH values 5.2, 6.1 and 5.0, respectively. Name the functional group associated with each of the three matrices, as well as the charge present under the operating conditions
Ans: Functional group for CMcellulose: carboxymethyl functional group -CH2OCH2COOH, Ph 5.2 weakly –ve, or weak cat ion exchanger CH2OCH2COO – below or equal to 5 its neutral
Functional group for Q-Sepharose: quarternary amine with a strongly positive charge at alkaline ph, at ph 6.1 its cation and strong anion exchanger.
SP-Sepharose: sulphonyl group, strong cation exchanger (-SO3¯), at ph 5 its –vely charged.
1c. which matrices do you think will be effective in adsorbing the protein with the buffers utilized. Justify your answer.
For most of the total protein adsorption CM-cellulose is the best column because it is weakly cation exchanger. Then Q-Sepharose captures most of the –vely charged proteins and eluted proteins mostly contain +vely chrged proteins. As our ph range is acidic the enzyme is positively charged and acidic hence most of the enzyme captures by SP-sepharose, hence this is the best.
Q1d. Suggest a pI range likely for the protein. Justify your answer (show your working) with respect to the different buffer and matrix chemistries utilized.
In the CM cellulose column CM2 active fraction eluting at 0.15M Nacl this means most of the proteins are not strongly positively charged, this means their pi is within acidic range. In Q-Sepharose also same which substantiates its pi to be in acidic range. In the SP-Sepharose column majority active fraction protein is eluting with 50mM NaCl Buffer means proteins with weak ionic interactions elute at low salt concentration, here protein should be weakly positive, this means its pI should be in the acidic range near the pH 5.
Q1e.Suggest a pI range likely for proteins eluting in fraction Q3. Justify your answer (show your working) with respect to the buffer and matrix chemistries utilized.
Q-Sepharose strongly +ve column and Q3 eleuting at 1M NaCl fraction strongly –vely charged proteins should elute here, hence their pi should be near neutral or in the alkaline range
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