lab 2 - enzymes I Flashcards
what do enzymes do
increase the rate of biochem reactions by lowering the activation energy required for the reaction to occur
what is the catalytic effect of the enzyme determined by
shape of active site
what does the rate of enzyme catalysed reaction depend on
concen of enzyme and substrate
enzyme sensitivity to high temp or extreme pH
presence of regulatory compounds that alter activity
what is enzyme kinetics
how fast an enzyme converts substrate to products and investigating the effects of varying the conditions of a reaction
what is a key parameter of enzyme kinetics
enzyme activity (rate or velocity of the reaction catalysed by the enzyme)
how can enzyme activity be measured
spectrophotometer
what do spectrophotometers measure
amount of light absorbed by the solution (absorbance) relative to how much light passes through the solution (transmittance)
absorbance of light by a molecule
what does the absorbance reading on a spectrophotometer tell us
how much light of the selected wavelength a particular pigment molecule absorbs
low value = low amounts of molecule
increase in value = increase in molecule concen
what can you find from the absorbance value when put on a standard curve
the concen of the solution that had the absorbance value
what is a standard curve
displays the relationship between absorbance and concentration for that pigment
what enzyme was used in lab 2
b-galactosidase
present in lactaid and used to treat lactose intolerance
what is lactose
disaccharide found in dairy
consists of glucose and galactose linked together by b-(1,4) glycosidic bond
(can be broken by b-galactosidase)
how do levels of b-galactosidase change through life
high levels at birth
adequate levels through life in europeans
decrease in asians and indigenous
what is the problem with measuring b-galactosidase
neither the natural substrate or products absorb light
what substrate is used
ONPG
- consists of galactose and o-nitrophenol with same link found in lactose
what happens when bond in ONPG is broken by b-galactosidase
produces galactose and o-nitrophenol (yellow - absorbance can be measured)
how do we produce a more pigmented end product
expose o-nitrophenol to alkaline pH (>8) converting it to o-nitrophenolate
intense yellow colour
how to measure b-galactosidase activity
enzyme solution is mixed with buffer designed to keep pH constant
substrate ONPG is added
solution is incubated at specific temp
b-galactosidase breaks down ONPG and forms o-nitrophenol
alkaline solution added - inactivates the enzyme and stops the hydrolysis of ONPG
what is a dilution
ratio of the volume of the original solution to the total volume of the diluted sample
D = V original solution / (V original solution + V dilutant)
how to find concen of dilution
diluted concen = original concen x dilution
how to calculate fixed volumes of specific concens from stock solutions
V1C1 = V2C2
1 = original
2 = new solution
what does the blank tube for the spectrophotometer contain
everything except pigment molecule
