19. You are a summer intern in a clinical hematology lab. The lab director gives
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Question
19. You are a summer intern in a clinical hematology lab. The lab director gives
you a sample of a patient’s blood proteins and asks you to characterize the
thrombin in the sample. She also tells you that thrombin is a serine protease
important in blood clotting (see Table 5.3), and this patient is a newborn
with uncontrolled bleeding.
(a) To characterize the thrombin in the sample, you must remove two
proteins that interfere with the thrombin activity assay: cytochrome c
and lactoglobin. You find some CM-cellulose (see Figure 5A.5) and a
phosphate buffer (pH = 6.4) on the shelf in your lab. You decide to load
the protein sample onto a column of CM-cellulose equilibrated in the
pH = 6.4 buffer. Predict the order of elution for the three proteins shown
in the table below. At pH = 6.4, which protein(s) do you predict will
remain bound to the column?
Protein
(b) List two different ways you could change the buffer to elute the bound
protein(s) and achieve proper separation of the proteins.
(c) You are surprised to observe that the patient’s thrombin flows through the
CM-cellulose column at pH = 6.4, and does not bind. Confident in your
technique, you suspect the patient’s thrombin is different from wild-type
thrombin. Using a different buffer system, you manage to purify some of
the patient’s thrombin and you submit the purified sample for amino acid
sequencing. The sequence analysis shows that the patient’s thrombin contains
a mutation in the enzyme active site. A lysine residue in the wild type
has been mutated to an asparagine in the patient’s thrombin. Does this mutation
explain the anomalous CM-cellulose binding behavior you observed?
Protein
pI Cytochrome c 10.6 Lactoglobin 5.2 Thrombin (wild type) 7.1 TABLE 5.3 The sequence specificities of some proteolytic enzymes and cyanogen bromide N-terrminal-N-C-C-N-C-CC-terminal H..H Enzyme Trypsin Chymotrypsin Thrombin V-8 protease Prolyl endopeptidase RPro Subtilisin Carboxypeptidase A R2 C-terminal amino acid From digestive systems of animals Thermolysin Cyanogen bromide Preferred Site R1 Lys, Arg R,-Tyr, Trp, Phe, Leu R1 Arg Source From digestive systems of animals, many other sources Same as trypsin From blood; involved in coagulation From Staphylococcus adireus Lamb kidney, other tissuès From various bacilli | R! = Asp, Glu Very little specificity R2Leu, Val, lle, Met R1 Met From Bacilus thermoproteolyticus The residues indicated are those next to which cleavage is most likely. In some cases, proforence is determined by the residue on tho N-tominal side of the cleaved bond (R.) and sometimes by the residue to the C-terminal sido (Re) Ganerally, proteases do not cleave whoro prolino is on the other side of the bond. Even prolyl andopaptidase wil not cleave R2-Pro. FIGURE 5A.5 The chemical structures of commonly used ion exchange media Weak anion exchanger (DEAE) Strong anion exchanger 'Q') (X)n (X)n Weak cation exchanger (CM) Strong cation exchanger ('S)Explanation / Answer
Thrombin is an important enzyme in humans that act as coagulant in human body. Thrombin is the serine protease that convert soluble fibronogen into insoluble fibrin. It help in blood clot.fibrinogen comprises 7% of blood protein and its conversion into fibrin is essential for blood clot.
Prothrombin becomes activated to serine protease to allow clot in blood during an injury. thrombin also promote platelet activation through activation of protease activated receptor.. Active thrombin was produced from purified prothrombin using several different method like coagulation protein.
To characterise its presence in human body, plasma is used as a starting sample and requires 12 ml of whole blood. Blood is collected in 60 ml antocoagulant citrate dextrose solution A.
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