enzyme assays

in an enzyme assay what are you trying to measure

-which is easier to measure

rate of the reaction by measuring the reate of the disappearance of substrate or the appearance of product

-easier to measure the appearance of a product

how is the amount of enzyme expressed

in terms of activity rather than the conc of the nezyme

what is the international unit for one unit of activyt

the amount of the enzyme that converts 1 micromole of substrate to product per minute at 25 degrees

what does one unit of activity measure

the rate of the reaction NOT how much of the enzyme you need

define the specific activity of an enzyme

the number of units of enzyme activity present per mg of protein

what does the specific activity tell you

the weight of the enzyme needed

explain how a typical catalysis assay is carried out

-add all components to a suitable vessel except one component

-when t=0 add a small volume of the missing component

-mix the vessel but not vigourously

-when using spectrophotometric assays the reaction may be started in the cuvette

what must you include in a typical assay

a blank must be included

what is a blank

contains all of the components except for the enzyme or the substarte

how can you ensure that the assay is valid

The enzyme is present in very small amounts relative to the other components

The initial velocity is directly proportional to [E]

The assay period must be short so that the concentration of substrate doesn't change too much and not much product accumulates

Blank rate is accounted for

pH is suitable and constant - pH optimum

Ionic strength is appropriate

Temperature is appropriate - Temperature optimum

why should you keep the enzyme at a low conc

-very expensive
-whole point of an enzyme is that you don't need that much and they can be reused

why must the assay period be short so

we dont want to much product accumulating as this may lead to an equilibrium and may distort the reading

-we only want to measure the initial burst

why must the PH be optimium

the wrong pH may cause the enzyme to become denatured

what are the 2 types of assays which can be preformed

continious and discontinious

what are continoius assays usually

spectrophotometric

what is a spectrophotometric assay

substrate or product absorbs light of specific wavelength

what do continious assays do

Monitor the rate of activity continuously

how do you calculate the rate in a continious assay

Calculate the rate using the extinction coefficient of the product (or substrate)

what occurs in discontionious assays

Samples are withdrawn at intervals, product is measured (by reference to a standard curve) and rate estimated.

this establishes a linear portion assay

in discontinuous assays what do you do with subsequent assays

measure once after a time that is within the linear portion

how can we use spectrophotometric assays to determine catalytic activity

-many dehydrogenase enzymes use cofactors such as NAD/NADH

-when NADH is used as a cofactor it is converted into NAD

-following this the amount of NADH decreases while the amount of NAD increases

-these two compunds absorb light at differnt wavelengths

-by shining different wavelengths on the sample we can determine how much of each is present at a certain time

-this can be then used to determine the rate of reaction

-if after sometime there is alot of NAD and not alot of NADH then we know that there is a reaction taking place

direct assay

direct measurement of substrate or product based on some property, absorbance, radioactivity, fluorescence

indirect assay

when assay is finished add a reagent that produces a measurable property

modified substance assay

• chemiluminescent

• fluorometric

• colorimetric

radiometric assay

radioactive assay is transferred from substrate to product

coupled assay

if you have a reaction you cannot measure easily couple the reaction to another one that produces measurable product