Task 3 23.11.2012
Mutant identification
Your first real task in biotechnological laboratories BTP (Biotechnology for purification) is
to distinguish between two mutant variants X1 and X2 of a protein which is used for the
decomposition of toxic substances in drinking water. Accidentally, labels of reaction
reservoirs with enzymes were confused. You have found out that both types of enzyme
contain one tryptophan. Additionally, you know that tryptophan in case of mutant X1 is
placed much closer to the surface and therefore it is in contact with surrounding solution
more than in the case of the second variant of protein X2.
The addressing of this task is important for the supply of drinking water to residents in an
area affected by drought. You can use your knowledge of fluorescence quenching for the
problem solution. You remember that the protein with tryptophan close to the surface can be
determined from the dependence of fluorescence intensity on the concentration of the
quencher. For quenching fluorophore, the basic Stern-Volmer equation can be applied:
where F0 is fluorescence intensity in the absence of quencher, F is the fluorescence intensity
in the presence of quencher, KSV is Stern-Volmer constant and [Q] is the concentration of the
quencher.
You have carried out measurements of fluorescence intensity of proteins taken from
reservoirs A and B. The fluorescence intensity was measured in the absence of the quencher.
Then you measured fluorescence drop after gradual addition of quencher (acrylamide). Your
obtained values of fluorescence intensity are in the table below.
Plot the dependence of relative fluorescence intensity decrease on the acrylamide
concentration in the form of Stern-Volmer graph and answer the following questions:
1. Is acrylamide dynamic or static quencher?
2. What are the constants KSV corresponding to each mutant variants of the
enzyme?
3. Determine in which reservoir the enzyme X1 is located.
Please send me your answers together with Stern-Volmer plot for A and B via email.
Correct answer = 0.5 point
][10
QK
F
F
SV
X1 X2
reservoir
Acrylamide concentration [M]
0 0.1 0.2 0.3 0.4 0.5
1 Bencúrová, Petra A 944 911 891 870 853 834
B 944 794 697 621 560 510
2 Dabravolski, Siarhei A 977 943 922 901 883 864
B 977 822 722 643 580 528
3 Dubec, Vít A 940 908 887 867 850 831
B 940 791 694 619 558 508
4 Dudová, Zdenka A 951 918 898 877 860 841
B 951 800 703 626 565 514
5 Dvořák, Jan A 960 927 907 886 868 849
B 960 808 709 632 570 519
6 Fabišik, Matej A 986 952 931 910 891 872
B 986 830 729 649 585 533
7 Fedorko, Jan A 938 906 886 865 848 829
B 938 790 693 617 557 507
8 Fialová, Martina A 957 924 903 882 865 846
B 957 805 707 630 568 517
9 Holek, Michal A 986 952 931 910 891 872
B 986 830 729 649 585 533
10 Kočka, Martin A 975 942 921 899 881 862
B 975 821 720 642 579 527
11 Míka, Matěj A 981 947 926 905 886 867
B 981 825 724 646 582 530
12 Obacz, Joanna
Agnieszka
A 957 924 903 882 865 846
B 957 805 707 630 568 517
13 Partyka, Jan A 936 904 884 864 846 828
B 936 788 692 616 556 506
14 Přikrylová, Terézia A 994 959 938 917 898 878
B 994 836 734 654 590 537
15 Rájecký, Michal A 984 950 929 908 890 870
B 984 829 727 648 584 532
16 Reichman, Pavel A 962 929 908 887 870 851
B 962 810 711 633 571 520
17 Sochorová, Jana A 964 931 910 889 871 852
B 964 811 712 635 572 521
18 Škubník, Karel A 953 920 900 879 861 842
B 953 802 704 627 566 515
19 Tylichová, Zuzana A 970 936 915 894 876 857
B 970 816 716 638 575 524