The purification table pasted below was copied from the following article Yang,
ID: 1048433 • Letter: T
Question
The purification table pasted below was copied from the following article Yang, C.-P; Fujita, A; Ashrafuzzaman, M. D; Nakamura, N; Hayashi, N. Purification and characterization of polyphenol oxidase from banana (Musa sapientum L.) pulp. 2000, 48, 2732-2735. • What type of protein properties were exploited in each purification step that followed after the crude extract was prepared? • Show how the final purification fold (635.7) and final recovery (3.0%) were calculated.
Table 1. Stepwise Purification Results of PPO of Banana Pulp volume total activity total protein pecific activity purification recovery (mL) (mg (fold) (unit) 388620 239592 158120 100317 32400 15360 11844 purification step (unit/mg protein) crude extract 80% (NH4)2SO4 saturation (crude enzyme) DEAD-Toyopearl 650M Butyl-Toyopearl 65OM Super Q-Toyopearl 650M Hydroxyapatite Toyopearl HW 55-S 6350 298 324 370 50 20 94 18732.5 2972.6 822.0 360.2 5.6 1.4 0.9 20.7 80.6 192.5 278.5 5785.7 10971.4 13160.0 1.0 3.9 9.3 13.4 279.5 530.0 635.7 100.0 61.7 40.7 25.8 8.3 4.0 3.0Explanation / Answer
After the crude extract was prepared,
i) Initially, precipitation is done to recover the protein from the crude extract, the property exploited
will be Solubility.
ii) DEAE-Toyopearl 650M is a weak anion exchanger (chromatography), the property exploited here
is Net charge.
iii) Butyl Toyopearl 650M is a hydrophobic interaction chromatography (HIC) resin for biomolecule
purification, the property exploited here is hydrophobicity of the surface.
iv) SuperQ Toyopearl 650M resin is a strong anion exchanger used for biomolecule purification,
the property exploited here will also be Net charge.
v) hydroxylapatite chromatography has been described as "mixed-mode" ion exchange,
where the properties exploited here will be adsorption & elution.
vi) Toyopearl-HW 55S is a size exclusion chromatography resin, therefore the properties exploited
here will be size and shape.
Purification fold:
specific-activity = total enzyme activity / total protein = 11844 / 0.9 = 13160
Purification fold = specific-activity / inital specific-activity = 13160 / 20.7 = 635.74879
Recovery % = (total enzyme activity / initial total activity)*100
recovery % = (11844 / 388620) *100 = 3.047 %
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