Lab Exam Flashcards
properties of enzymes
bind reactant (Substrate), alter its configuration so that it is more easily changed into the product, enzyme remains unchanged over the course of the reaction
enzyme and Ea
Ea: activation energy - energy barrier needed to overcome for a cellular reaction to occur
enzymes reduce the Ea for the reaction, and increase RR/velocities of the reaction.
- do not affect ∆G’º
- decrease Ea/∆G†
- speed up reversible reactions by the same degree in both directions
draw diagram
Michaelis-Mentin plot
define relationship between [S], V0 and Vmax
contestant Km = [S] when 1/2Vmax = V0
draw diagram
Km measures what?
measure of the efficiency of an enzyme, Km is the concentration of the substrate which produces a RR of half Vmax
a low Km (closer to zero) indicates high affinity of E for S
enzyme kinetics what does each value stand for
V0 = initial velocity or initial RR of enzyme r’n
Vmax: max velocity of enzyme r’n
Km = 1/2Vmax indicates efficiency of enzyme
steady state kinetics
kinetics of enzyme r’n after first few microseconds when [ES] and conc of any other intermediates remain constant,
usually applies to measurement of V0
lineweaver-burk plot (Double reciprocal)
y axis: 1/V0
x axis 1/[S]
y - axis intercept: 1/Vmax
x - axis intercept: -1/Km
can use y = mx + c to find values of graph, eg x = 0 to find y int
factors affecting protein structure
denaturation vs renaturation
pH, temp, solubility, ionic strength
den: loss of biological activity
ren: regains biological activity
enzyme inhibition
inhibitors: compounds that decrease enzymes activity
irreversible inhibitor
inactivator that reacts with the enzyme, permanently shut off the enzyme molecule.
often toxins, can be used as drugs
reversible inhibitor
2 ways of functioning
bind and dissociate from an enzyme, temporary inhibiton
often structural analogy of substrate or product, used as drugs to slow down a specific enzyme
- bind to free enzyme and prevent substrate from binding - temporarily, eventually dissolve and enzyme works again
- bind to the ES-complex and prevent reaction from occurring (Temporarily)
competitive inhibition
lines intersect on the y-axis
∆ 1/Km, same 1/Vmax
binds directly to AS, competing directly with substrate
uncompetitive inhibition
lines parallel across y-axis
dec 1/Km and dec 1/Vmax
binds to another part of enzyme (not AS) sits on allosteric site, allows substrate to bind but interferes with efficiency and RR
mixed inhibition
lines intersect before the y-axis
inc 1/Km, dec 1/Vmax
most effective, bind to AS, allosteric site, substrate or anything on enzyme and shut it off, decreases efficiency of enzyme and interferes with velocity / RR
noncompetitive inhibition
lines start together on -x-axis
same 1/Km, dec 1/Vmax
bind to allosteric site, doesn’t interfere with enzyme binding to substrate, interferes with velocity, and stops overall reaction
Bradfords assay:
shifts absorbance from 470nm to 595nm
intensity of blue correlates with concentration of proteins,
measured qualitatively with eye (compare colours) or quantitatively with spectrophometer
dye: Coomassie brilliant blue dye
limitations of Bradfords assay
measures total [protein], need further methods to identify specific proteins
assay is linear over limited range
dye binds directly to arginine and hydrophobic AA
AA composition can alter the concentration-absorbance curve
standard with a similar composition to unknown must be used - eg BSA
Beer’s Law
states that if a solute absorbs light of a particular wavelength, the absorbance is directly proportional to the concentration of that solute in solution up to a point
Beer’s law formula
A = kLc / A = Ebc
k - constant / molar absorptivity
L - light path length (~1cm)
c - concentration of compound in solution
qualitative vs quantitative determination of protein concentration
qualitative: visually compare the colour of unknown sample against standards of known concentration
quantitative: A595nm for each standard and generate standard curve with data, unknown sample into the linear equation to determine concentration
properties proteins can be separated by:
charge, size, affinity for a ligand, solubility, hydrophobicity, thermal stability
chromatography used for protein separation based on one of those properties
chromatography
distribution of compounds between 2 immiscible phases
- stationary phase and mobile phase
selection of the 2 immiscible phases is dependent on the characteristics of the molecules of interest
size exclusion chromatography
separation of proteins based on molecular size. large molecules too big for the bead pores eluted first, smaller molecules interact with bead pores and so elute later.
elution time/volume is inversely proportional to molecular weight (molecular weight not proportional to molecular size)
ion exchange chromatography
separation by charge. proteins charged due to phosphate group and electrostatically bind to opposite charges.
columns covalently bond charged molecules to the insoluble resin. eg with a cation exchanger, proteins with more negative net change move faster and elute earlier
ligand affinity chromatography
solution of ligand is added to column, specific to protein of interest. protein of interest eluted first by ligand solution.
electrophoresis
analytical separation of proteins. electrical field pulls/separates proteins according to charge. travel towards +ve terminal. gel matrix hinders mobility of proteins according to size and shape - eg only separated out by charge.
SDS-PAGE
used to calculate molecular weight of protein
sodium dodecyl sulfate - polyacrylamide gel electrophoresis
plasmids 3 essential parts
plasmid: circular piece of bacterial DNA
ori: origin of replication. DNA polymerase/other proteins required for DNA synthesis bind to the ori
Selectable marker: usually drug resistance. provides a way to select for bacteria that contain plasmid
MCS: multiple cloning site (not essential), region with several restriction enzyme recognition sequins the provide cloning sites for insertion of your gene of interest. MCS used to cut open because lots of palindromes are present
- 2nd fail safe: LacZ operon, gives bacteria availability to break down lactose into galactose and glucose - in the event that the B-galactosidase gene is disrupted
palindrome defn and fn
sequence of DNA that is the same when read in opposite directions (fwd and back) eg GAATTC is CTTAAG
restriction enzymes
specific endonuclease (cut inside DNA, as plasmids are circular, no need for exonuclease)
- recognise short sequence of NDA and cleave DNA at or near the recognition sequence (palindromes)
- cut into sticky or blunt ends (sticky ends can bind with other DNA molecules with same overhands).
serve as a natural defence mechanism for bacteria against viral infection, prevent replication if phage DNA by cutting it into too many pieces
DNA ligase fn
enzyme that joints the ends of DNA and re-establishes the phosphidester bond in the DNA molecule, puts sticky and blunt ends back together
