This document describes the practicals for cellular biochemistry. The first practical is gel filtration chromatography. Then a linked enzyme assay was performed to measure the activity of hexokinase to determine the glucose concentration in unknown samples. Finally, the electron transport chain act...
Practical One: Gel Filtration
How GF chromatography works: there is a stationary phase (Sephadex beads) and a mobile
phase (buffer). When you put buffer at the top, they push the molecules down, which pass the
stationary phase (beads kind of, with holes). Big ones do not enter the beads, they go straight
out. Smaller, takes more time, so they come after-like tutorial. This is called size exclusion
chromatography. Never let column dry-always pour more buffer! GF chromatography can do two
things: separate molecules according to size and used to determine the molecular weight of
molecules. Determination of the elution volumes/time of several proteins with known molecular
weights, allows us to make a calibration curve for the particular FD column.
Sephadex: G-75, water absorption is 7.5 ml/g dry mass, molecular weight fractional range is
3k-70k Da.
GF Column Parameters:
exclusion volume/void
volume (Vo) is volume
between gel beads.
Vo=elution volume of large
molecules that do not enter
the pores. For Sephadex
G-75, proteins with
molecular weights greater
than 70k are completely
excluded. Internal pore
volume (vi) is the volume
inside the beads. Vi is
volume of solvent/buffer
held in the pores. Elution
volume (ve) is the volume
required to elute any
particular molecule. Elution
volume is a general term.
Total volume is the total
volume of the mobile phase
in the column (Vt = Vo +
Vi). Also equal to the
amount of the buffer
required to run a substance
through the column (eluting
a substance) when the
substance is small enough
to completely penetrate the
pores of the gel. Kav
coefficient: (Ve-Vo)/(Vt-Vo); it is a ratio. It is easy to get and more useful in practice (MW
determination of unknown protein). For very large molecules eluting at Ve = Vo, = 0. For small
molecules eluting at Ve=Vt, then = 1. Because is coefficient, is always between 0 and 1.
, Aim: separate a complex mixture of proteins using GF chromatography and determine the
molecular weight of alpha-amylase using GF chromatography. Key Facts: Alpha amylase is not
coloured; can be detected by its enzymatic activity-hydrolysis of starch. Dextran blue,
hemoglobin, cytochrome c, riboflavin are coloured compounds.
Enzymes need to be at
cool temperature or they lose their activity. Did contain? Is yellow orange clear then?
Temperature effect on alpha amylase activity: 0 degrees, no digestion. 10 degrees, 12 minutes
there is one clear (alpha does hydrolyse starch) and red (little bit), 20, 8 minutes there is lots of
hydrolyse. 30 (5 min) and 40 (3 min) lots of hydrolyse! 50 though, 10 minutes and goes back to
what 20 looked like. Based on this, optimum temperature is 38 or so. Iodine turns blue-black if
starch is present; when iodine no longer changes colour, there is no starch present. 60 degree,
no digestion: high temp, the enzymes do not work.
Results & Discussion
We did an enzyme assay to find alpha amylase’s molecular weight.
Practical Two: Linked Enzyme Assay
Going to practice enzyme kinetics and see reality. Most enzyme assays monitor the
disappearance of a substrate or appearance of product.
Linked/coupled enzyme assay: if neither the substrates nor the products of an
enzyme-catalysed reaction absorb light at an appropriate wavelength, the enzyme can be
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