A crude cell extract containing 2 g.L -1 of protein was assayed for enzyme activity using...
A crude cell extract containing 2 g.L -1 of protein was assayed for enzyme activity using an excess of substrate. It was found that 5 ml of the solution converted 25 µmoles of substrate to product over a period of 2 minutes.
(i) Calculate the specific activity of the enzyme.
The crude extract was subjected to anion exchange chromatography followed by gel filtration. After anion exchange the specific activity had risen to 226 µmoles.min-1 .mg-1 , and after gel filtration it rose further to 267 µmoles min-1 mg-1 .
(ii) Calculate the degree of purification achieved at each stage
(iii) What additional pieces of information would you need to calculate the percentage recovery after each step?
Homework Answers
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A crude cell extract containing 2 g.L -1 of protein was assayed
for enzyme activity using...
Sample Problem 1 TABLE 3-5 A Purification Table for a Hypothetical Enzyme Fraction volume (ml) Total...
Sample Problem 1 TABLE 3-5 A Purification Table for a Hypothetical Enzyme Fraction volume (ml) Total protein (mg) Activity (units) Procedure or step Specific activity (units/mg) 1,400 1. Crude cellular extract 2. Precipitation with ammonium sulfate 3. Ion-exchange chromatography 4. Size-exclusion chromatography 5. Affinity chromatography 10,000 3,000 400 10 32 100,000 96,000 80,000 60,000 45,000 200 600 15,000 • If affinity chromatography is the final step of purification, what is the yield and fold purification. Protein X has an absorptivity...
You are to purify a highly soluble homotetrameric protein containing a NADP binding site, in its...
You are to purify a highly soluble homotetrameric protein containing a NADP binding site, in its functional form, and a pI = 9 with a subunit molecular weight of 35,000 Da from a crude extract of E. coli. For this protein, select the four best techniques from the list below and place them in the correct order for the most reasonable strategy to obtain homogeneous protein. (A) Precipitation of proteins using high ionic strength (40%) ammonium sulfate, followed by a...
The specific activity of a pure enzyme is 6000 μmol substrate transformed.min-1.(mg protein)-1 and its molecular...
The specific activity of a pure enzyme is 6000 μmol substrate transformed.min-1.(mg protein)-1 and its molecular weight is 10,000 Da. Calculate the turnover number of the enzyme in units of μmol substrate transformed.(μmol enzyme)-1.sec-1]? Show all workings including units. Note: A calculator should not be required to answer this question.
Based on the results in the table, is anion exchange a good purification step, yes or...
Based on the results in the table, is anion exchange a good
purification step, yes or no. Defend your answer. Could someone
please explain how you would know if the purification worked
looking at a table like this? Which values would change?
Dous lloded during the way to Proteins in eine Wrn that once the non-poc b. A student is purifying an enzyme wid heyme which catályzes the Oxidation of alcohol functional groups Gising NAD as a cofactor. NADH t...
a-d plz 7. Protein purification. As a graduate student, the first protein I purified was RNA...
a-d plz
7. Protein purification. As a graduate student, the first protein I purified was RNA polymerase (RNAP) from E. coli- Some physical and chemical properties of Ecoli RNAP Molecular max470,000 g/mol polynentide compasitian (subunits): (50 kDa), k andok a pl = 5.34 substrates: NTPs cofactor: Mg? Purification protocol. E coli cells were broken usine Isozyme, yielding a cellualar extract containing a protcome solution 4M (NHOSO, was added to the cellular extract. A white protein precipitate was formed incuding RNAM...
Question 3. Draw the tripeptide: Val-Ser-Ala. Include a phosphoryl group on the amino acid sidechain that...
Question 3. Draw the tripeptide: Val-Ser-Ala. Include a phosphoryl group on the amino acid sidechain that is most likely to undergo phosphorylation. Question 4. A protein retained on an ion exchange chromatography column is usually eluted off the column by (circle the correct answer) A. increasing the salt concentration in the buffer B. adding the protein's free ligand. C. changing the temperature of the elution buffer. D. allowing the retained protein to naturally come off the column after the non-specifically...
SIRJE GEL OBJECTIVE: Partial purification of a protein, isocitrate dehydrogenase (IDH), using DEAE ion exchange chromatography....
SIRJE GEL OBJECTIVE: Partial purification of a protein, isocitrate dehydrogenase (IDH), using DEAE ion exchange chromatography. IDH will be eluted from the column using increasing concentrations of KCI. IDH will be tracked using the IDH activity assay and the extent of purification will be analyzed using SDS PAGE Last week we assayed for the activity of the enzyme, isocitrate dehydrogenase, from E.coli strains carrying a plasmid that over-expresses the E. coli IDH gene. To purify proteins from cel extracts, biochemists...
1.) Describe the goals of a Gel Filtration Chromotography Experiment??? 2.) Explain each key theoretical principle...
1.) Describe the goals of a Gel Filtration Chromotography
Experiment???
2.) Explain each key theoretical principle of a Gel Filtration
Chromotography, and how they help acheive the goal???.
4.) Explain the key equations used in the Gel Filtration
Chromotography experiment and the terms involved in the
equation????
HI im trying to prepare for a lab/report and i have some
questions i could use help with please :) over all having trouble
seeing how everything ties together etc :) thank you...
Cyw Ala Ατα Ile Ausn Lou Asp aly Lys Ser Which molecule below is asubunit of...
Cyw Ala Ατα Ile Ausn Lou Asp aly Lys Ser Which molecule below is asubunit of peptide chains a. Acetic acid b. Alanine O. ATP d. Gracil Report the charge on most aqueous aspartie acid molecules at pH-7. Report the approximate p values for the amino acid whose titration curve is shown below (note there may be more slots for answer than answers). A) Value 1 B) Value 2 c) Value 3 c) Value 4 The structure of a rare...
PREPARATION OF PHARMACEUTICALS - Fischer Esterification Reactions Q: Calculate the expected mass of both methyl salicylate and aspirin assuming 100% yield. EXPERIMENT7 PREPARATION OF PHARMACEUTICALS...
PREPARATION OF PHARMACEUTICALS - Fischer Esterification
Reactions
Q: Calculate the expected mass of both methyl salicylate and
aspirin assuming 100% yield.
EXPERIMENT7 PREPARATION OF PHARMACEUTICALS Fischer Esterification Reactions Almost 2500 years ago, physicians such as Hippocrates recommended that patients chew on the bark of the willow to alleviate pain. The active ingredient in willow bark was found to be salicin, a compound made of a molecule of salicyl alcohol bonded to a p-D-glucose molecule. In the stomach, the bond between...
Sample Problem 1 TABLE 3-5 A Purification Table for a Hypothetical Enzyme Fraction volume (ml) Total protein (mg) Activity (units) Procedure or step Specific activity (units/mg) 1,400 1. Crude cellular extract 2. Precipitation with ammonium sulfate 3. Ion-exchange chromatography 4. Size-exclusion chromatography 5. Affinity chromatography 10,000 3,000 400 10 32 100,000 96,000 80,000 60,000 45,000 200 600 15,000 • If affinity chromatography is the final step of purification, what is the yield and fold purification. Protein X has an absorptivity...
Based on the results in the table, is anion exchange a good
purification step, yes or no. Defend your answer. Could someone
please explain how you would know if the purification worked
looking at a table like this? Which values would change?
Dous lloded during the way to Proteins in eine Wrn that once the non-poc b. A student is purifying an enzyme wid heyme which catályzes the Oxidation of alcohol functional groups Gising NAD as a cofactor. NADH t...
a-d plz
7. Protein purification. As a graduate student, the first protein I purified was RNA polymerase (RNAP) from E. coli- Some physical and chemical properties of Ecoli RNAP Molecular max470,000 g/mol polynentide compasitian (subunits): (50 kDa), k andok a pl = 5.34 substrates: NTPs cofactor: Mg? Purification protocol. E coli cells were broken usine Isozyme, yielding a cellualar extract containing a protcome solution 4M (NHOSO, was added to the cellular extract. A white protein precipitate was formed incuding RNAM...
Question 3. Draw the tripeptide: Val-Ser-Ala. Include a phosphoryl group on the amino acid sidechain that is most likely to undergo phosphorylation. Question 4. A protein retained on an ion exchange chromatography column is usually eluted off the column by (circle the correct answer) A. increasing the salt concentration in the buffer B. adding the protein's free ligand. C. changing the temperature of the elution buffer. D. allowing the retained protein to naturally come off the column after the non-specifically...
SIRJE GEL OBJECTIVE: Partial purification of a protein, isocitrate dehydrogenase (IDH), using DEAE ion exchange chromatography. IDH will be eluted from the column using increasing concentrations of KCI. IDH will be tracked using the IDH activity assay and the extent of purification will be analyzed using SDS PAGE Last week we assayed for the activity of the enzyme, isocitrate dehydrogenase, from E.coli strains carrying a plasmid that over-expresses the E. coli IDH gene. To purify proteins from cel extracts, biochemists...
1.) Describe the goals of a Gel Filtration Chromotography
Experiment???
2.) Explain each key theoretical principle of a Gel Filtration
Chromotography, and how they help acheive the goal???.
4.) Explain the key equations used in the Gel Filtration
Chromotography experiment and the terms involved in the
equation????
HI im trying to prepare for a lab/report and i have some
questions i could use help with please :) over all having trouble
seeing how everything ties together etc :) thank you...
Cyw Ala Ατα Ile Ausn Lou Asp aly Lys Ser Which molecule below is asubunit of peptide chains a. Acetic acid b. Alanine O. ATP d. Gracil Report the charge on most aqueous aspartie acid molecules at pH-7. Report the approximate p values for the amino acid whose titration curve is shown below (note there may be more slots for answer than answers). A) Value 1 B) Value 2 c) Value 3 c) Value 4 The structure of a rare...
PREPARATION OF PHARMACEUTICALS - Fischer Esterification
Reactions
Q: Calculate the expected mass of both methyl salicylate and
aspirin assuming 100% yield.
EXPERIMENT7 PREPARATION OF PHARMACEUTICALS Fischer Esterification Reactions Almost 2500 years ago, physicians such as Hippocrates recommended that patients chew on the bark of the willow to alleviate pain. The active ingredient in willow bark was found to be salicin, a compound made of a molecule of salicyl alcohol bonded to a p-D-glucose molecule. In the stomach, the bond between...
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