Analytical methods and enzymolgy
What is the uniqueness of the method of studying of analytical methods and enzymology that you offer?
The offered course includes the enhancement of knowledge in the given
field in several stages. Due to the way the kits are assembled,
every successive stage is an addition to the previous stage,
and every previous stage on the other hand is the basis of
the information of the next stage. The enzymes and other biologically
active compounds (BAC) are included in concentrations that
are necessary to conduct different lab practices.
The end result of these works are dependency graphs of different sizes.
These graphs are constructed given the data obtained by the experimenter,
and they must be situated within the limits necessary for drawing
different conclusions. That, in turn, is achieved by the correct dosages
of BAC contained in the kits offered to you.
1) Purpose of the Semester
To teach the students to model and determine the activity of enzymes
using the invertase as an example
2) Aside from optimums T (temperature) and pH (acidity) what other information
is needed for modeling enzyme activity?
Aside from optimums T (temperature) and pH (acidity) that are preceived
as known, it is critical to determine the concectration of substrate for
3) How to determine the concentration of [S] substrate?
Substrate concentration [S] = 10Km, where Km is the Michaelis constant.
4) How to determine the Michaelis Km constant?
Km is determined from the dependence of maximum speed of reaction Vmax
and substrate [S] concentration. The speed of the reaction is determined
by measuring the concenration of glucose produced over time.
5) How to determine glucose concentration?
Glucose concentration is determined from glucose calibration curve.
6) How to construct glucose calibration curve?
Glucose calibration curve is constructed from dependencies of standard
solutions of glucose and their optical densities. Thus the overall activity
is calculated. Determining specific activity is especially important.
7) How to calculate specific activity of the enzyme?
For determining special activity, it is necessary to determine protein
concentration which is calculated from the protein calibration curve.
Relationship of values of overall activity to the protein concentration
give the specific activity value.
8) How to construct protein calibration curve?
Protein calibration curve is built from dependencies of standard solutions
of protein and their optical densities. From this dependency, the protein
concentration is calculated.
What is the uniqueness of the method of studying of ion-exhange chromatography that you offer?
The absorbent (protein) is pre-stained, and when adsorbing,
it changes the color of the adsorbent from white to blue. In the process
of chromatography during the increase in ionic strength the protein is
desorbed off the column by using unique
technology. The location of desorbtion is colored
red - that is, it is possible to visually observe
how and where the protein desorbed, and when and how
it is washed off. Besides, we use
a pre-stained marker, that is visually observed in the process of
separation, therefore eliminating the need for additional
equipment for the detection of separating components.
Gel filtartion chromatography
What is the uniqueness of the method of studying of gel filtration chromatography that you offer?
Classically, the gel-filtration is conducted on a column sized 1-1.5m.
The carrier of type SEPHADEX is used, which separates proteins
in different ranges, although for this separation, it requires time from
10 hours to 3 days. For detection, it is also necessary to have
an ultraviolet detector, a self-recorder and a collector.
In the kits we offer, a colon sized 27 cm is used, as well as
a polymer, designed especially to have the ability to separate molecules
within 40 minutes on a column of this size.
Thin layer chromatography
What is the uniqueness of the method of studying of thin layer chromatography that you offer?
In the method we offer, pre-stained markers are used.
They can separate on short distances. In the process of
chromatography, it is possible to observe how the separation
into separate components from the injected solution takes place.