(a) A solution containing these five proteins was adjusted to pH 7.0 and then applied to a SIZE-EXCLUSION COLUMN. Assume that these five protein molecules are spherical in shape. Which protein is likely to be eluted LAST from the column? Explain briefly. (b) In another experiment, this protein mixture was first adjusted to pH 6.7 before applying to a CATION-EXCHANGE COLUMN. (i)What is the net charge on phosphorylase b? (ii) Which of these five proteins will bind to the CATION-EXCHANGER?
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The following table shows the molecular masses (Mr) and isoelectric points (pI) of five proteins:
Protein |
Mr |
pI |
Chicken egg white lysozyme |
14,000 |
11.0 |
Carbonic anhydrase |
30,000 |
5.4 |
Ovalbumin |
45,000 |
4.5 |
Phosphorylase b |
97,000 |
6.7 |
Lactate dehydrogenase |
140,000 |
7.12 |
(a) A solution containing these five proteins was adjusted to pH 7.0 and then applied to a SIZE-EXCLUSION COLUMN. Assume that these five protein molecules are spherical in shape. Which protein is likely to be eluted LAST from the column? Explain briefly.
(b) In another experiment, this protein mixture was first adjusted to pH 6.7 before applying to a CATION-EXCHANGE COLUMN.
(i)What is the net charge on phosphorylase b?
(ii) Which of these five proteins will bind to the CATION-EXCHANGER? Explain briefly.v
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- We want to measure the activity of alanine aminotransferase (ALAT) present in a serum. The reaction catalyzed by the enzyme is: Reaction 1: I- *H₂N- glutamate H - C-COO CH₂ CH₂ COO 0.1 M phosphate buffer pH 7.4 : 550 μL 1.2 M alanine: 100 μL CH3 pyruvate CH3 C time (min) A340 CIO O COO The enzyme reaction is alized in the following conditions: In a 1 cm-cuvette are added: COO™ pyruvate lactate dehydrogenase* (LDH, 300 µμg.mL-¹): 50 μL 1.5 mM NADH : 200 μL 0.04 M a-ketoglutarate: 500 µL serum containing ALAT: 600 μµL ALAT NADH + H+ 0 0.915 a-cétoglutarate COO LDH * Lactate dehydrogenase (LDH) reduces pyruvate into lactate, with the concomitant oxydation of NADH. This allows to indirectly measure the amount of product formed. с=0 CH₂ 1 0.741 Reaction 2: NAD+ CH₂ COO™ H-C CH3 OH COO™ lactate alanine H + *H3N-C The reaction is performed at 25 °C and the absorbance at 340 nm is monitored every minute, for 5 min. The absorbance values are given in the table below: Data: ENADH at 340 nm =…We want to measure the activity of alanine aminotransferase (ALAT) present in a serum. The reaction catalyzed by the enzyme is: Reaction 1: +H3N- glutamate H C CH₂ CH₂ COO -COO + pyruvate CH3 C=0 0.1 M phosphate buffer pH 7.4 : 550 µL 1.2 M alanine : 100 μL CH3 time (min) A340 COO COO™ pyruvate lactate dehydrogenase* (LDH, 300 µg.mL-¹): 50 μL 1.5 mM NADH: 200 μL 0.04 M a-ketoglutarate: 500 μL serum containing ALAT: 600 μL The enzyme reaction is realized in the following conditions: In a 1 cm-cuvette are added: 0 0.915 ALAT NADH + H+ LDH a-cétoglutarate COO * Lactate dehydrogenase (LDH) reduces pyruvate into lactate, with the concomitant oxydation of NADH. This allows to indirectly measure the amount of product formed. Reaction 2: NAD+ 1 0.741 C=O H CH₂ CH₂ COO™ CH3 C-OH COO lactate The reaction is performed at 25 °C and the absorbance at 340 nm is monitored every minute, for 5 min. The absorbance values are given in the table below: Data: alanine ENADH at 340 nm = 6220 M¹.cm1. One…Consider the proteins in the table below. Protein Isoelectric point (pl) М.W. Alcohol dehydrogenase 150,000 5.5 Cytochrome C 12,400 10.5 Serum albumin 66,000 5.3 B-Amylase 200,000 5.7 Apoferitin 443,000 4.0 Fill in the blank. If the mixture of proteins above was subjected to anion exchange chromatography at pH 8, from the column. would be the first protein to elute
- You run 5 standard proteins listed below on a size-exclusion (gel filtration) column with limit of 200,000Da. Please draw a chromatogram on a separate page with each peak and both axes labeled. Protein B-Amylase Alcohol dehydrogenase Bovine albumin Carbonic anhydrase Cytochome A280 MW 223,800 0.5 82,000 0.6 66,463 0.45 30,000 0.43 12,000 0.8Acid phosphatases are an important group of enzymes that can be detected in human blood serum. Under slightly acidic conditions (pH 5.0), this group of enzymes can hydrolyze biological phosphate esters as follows in the picture Acid phosphatases are produced and can be detected in erythrocytes, kidney, spleen, the liver, and prostate gland. The enzyme from the prostate gland is clinically important because an increased activity in the blood is frequently an indication of cancer of the prostate gland.Tartrate ion can strongly inhibit the phosphatase from the prostate gland, but not acid phosphatases from other tissues. How can you use the information above to develop a specific procedure for measuring the activity of the acid phosphatase of the prostate gland in human blood serum?H CH₂ H₂C HC-CH3 CH₂ H H₂C (S) H₂C H CH₂ CH₂ CH₂ NH O C NH NH₂ a) Which of the following statements about this peptide are correct? Group of answer choices Treatment of this peptide with trypsin generates two products. This peptide is a substrate for carboxypeptidase A Treatment of this peptide with cyanogen bromide generates a pentapeptide and a tripeptide. Treatment of this peptide with chymotrypsin generates three products. Treatment of this peptide with elastase generates 2 products. None of the above statements are correct. b) What is the sequence of this peptide using one letter abbreviations? c) What is the pH which would correspond to the ionization of the peptide as drawn above? 1, 5, 7, 10, 14
- On the right is a diagram of plasma concentration of three metabolites: glucose, B- hydroxybutyrate, and free fatty acid of an experimental animal over weeks of starvation. 4. (a) Label the lines with the names of the three metabolites. Yellow: Red: 10 20 30 40 Blue: Days of starvation (b) What factor in the plasma binds to and transports free fatty acid (c) Where was B-hydroxybutyrate produced? Why it is produced? What are the purposes of producing it (d) How B-hydroxybutyrate is used as fuel in extrahepatic cells? Write down the reactions, enzymes, and cofactors (No need to elaborate on the TCA cycle). (e) How many ATP molecules or equivalent are generated after complete oxidation of one molecule of B-hydroxybutyrate? Show your calculations. Hint: You need to consider ATP consumption for bypassed steps that may normally yield or consume AT Plasma concentration (mM)Acid phosphatases are an important group of enzymes that can be detected in human blood serum. Under slightly acidic conditions (pH 5.0), this group of enzymes can hydrolyze biological phosphate esters as follows: R-O-P-O;² + H 20 → R-OH + HO-P-O;² Acid phosphatases are produced and can be detected in erythrocytes, kidney, spleen, the liver, and prostrate gland. The enzyme from the prostrate gland is clinically important because an increased activity in the blood is frequently an indication of cancer of the prostrate gland. Tartrate ion can strongly inhibit the phosphatase from the prostrate gland, but not acid phosphatases from other tissues. How can you use the information above to develop a specificHemoglobin molecules exposed to high levels of glucose areconverted to glycated products. The most common, referred to as hemoglobin A1C (HbA1C), contains a b-chain glycatedadduct. Because red blood cells last about 3 months, HbA1Cconcentration is a useful measure of a patient’s blood sugarcontrol. In general terms, describe why and how HbA1Cforms.
- The proteins given below are separated using CM cellulose at pH 7.0 Write the order of elution of these proteins from the column Proteins pl Fibrinogen 5.8 Hemoglobin 7.1 Lysozyme 11.0 Pepsin 1.0 Ribonuclease 7.8Homocystinuria is caused by a defect in cystathionine beta-synthase (or 13-synthase), which leads to an accumulation of homocysteine in the blood. This accumulation causes symptoms such as a tall, thin frame, flushed cheeks, and osteoporosis (thinning of the bones). These individuals should limit their intake of proteins that contain methionine, such as egg whites. Using your understanding of biochemistry, explain why people with Homocystinuria should not consume egg whites and other such proteins.Acid phosphatases are an important group of enzymes that can be detected in human blood serum. Under slightly acidic conditions (pH 5.0), this group of enzymes can hydrolyze biological phosphate esters.Acid phosphatases are produced and can be detected in erythrocytes, kidney, spleen, the liver, and prostrate gland. The enzyme from the prostrate gland is clinically important because an increased activity in the blood is frequently an indication of cancer of the prostrate gland. Tartrate ion can strongly inhibit the phosphatase from the prostrate gland, but not acid phosphatases from other tissues. How can you use the information above to develop a specific procedure for measuring the activity of the acid phosphatase of the prostrate gland in human blood serum?