Lab 4 Flashcards
Define Enzymes
enzymes are proteins that act as catalysts in specific biochemical reactions, including the metabolism of drugs
explain the fate of drugs that are metabolized
they have undergone a chemical change that will dictate their pharmacological and toxicological profiles
true or false
enzymes involved in drug metabolism also metabolize endogenous substances such as hormones, vitamins, and lipids
TRUE
_____ is the primary site of metabolism for most drugs
the liver
as mentioned, the liver is the primary site of metabolism for most drugs.
name other significant organs for drug metabolism
kidneys
lungs
GI tract
blood
nervous tissue
skin
define first pass metabolism
the ability of the liver (and extrahepatic tissues) to metabolize drugs before they reach systemic blood circulation
where are cytochrome P450 enzymes found?
in liver cells in the membrane of the endoplasmic reticulum
name 2 examples of endogenous molecules
nutrients and hormones
name 2 examples of exogenous molecules
drugs and xenobiotics
the metabolism of both endogenous and exogenous molecules are classified as…..
phase 1 and phase 2
do phase 1 and phase 2 metabolism occur in most tissues?
yes (liver, kidney, skin, and lungs)
but the LIVER is the PRINCIPAL SITE for DRUG METABOLISM
the primary purpose of liver metabolism is __________
drug inactivation aka detoxification
the primary purpose of liver metabolism is drug inactivation (detoxification)
what is a concern with this
some drug metabolites ARE pharmacologically active and sometimes may even be more potent than the parent compound
Prodrugs are designed for….
conversion to an active moiety in the liver
what can you say about molecules that are very lipophilic?
they need to be converted to a hydrophilic molecule to facilitate their excretion and/or to increase their reactivity with cellular macromolecules such as protein and DNA
TRUE OR FALSE
drugs always undergo phase 1 and phase 2 of metabolism
FALSE
they may only undergo phase 1 or 2 depending on their functional groups and polarity
what does the S9 fraction contain?
both phase 1 and phase 2 drug-metabolizing enzymes
phase 1 = cytochrome P450 in the microsomes
phase 2 = cytosolic fraction
_______ is a valuable tool to study the in vitro metabolism of drugs.
explain
S9 fraction bc it contains both phase 1 (cytochrome P450 in the microsomes) and phase 2 (cytosolic enzymes) drug metabolizing enzymes
how can you predict the metabolic fate of the compound of interest? (drug)
by incubating it with the S9 fraction
the quality of the S9 fraction and other enzyme extracts is generally based on……..
the enzymatic activity present in 1mg of protein
when COMPARING enzyme extracts, the concentration of the protein in the extract must also be analyzed
true or false
there are few types of assays one can use to determine the protein concentration of a sample
FALSE – there are many types
as mentioned, there are many types of assays one can use to determine the protein concentration of a sample.
when deciding which type of assay, what 4 things should you take into account?
-how sensitive the assay is
-how difficult the assay is to run
-does the sample contain
compounds that interfere with the assay?
-how long the assay takes and expenses of reagents
How do you determine the concentration of an unknown sample?
a standard curve must be generated
comprised of data points of increasing known concentrations of proteins and their measured absorbance values. in protein assays, this trend will be linear
SO the equation of the trendline can be used to predict the concentration of your unkown
“the equation of the trendline of a standard curve can be used to determine the protein concentration of your unknown.”
what is the term for this?
interpolation
what can you sometimes do to ensure that the absorbance of the unknown falls within the standard curve absorbances
dilute your protein sample
what is extrapolation
calculating for protein values outside of the standard curve values
is there anything wrong with doing extrapolation?
this can result in error in calculating the protein concentration
explain how standard curves are usually plotted
Y-axis = absorbance
X-axis = protein concentration of the standards
what protein should be used for the standard curve?
why?
BSA (bovine serum albumin)
this is because it is a well-defined, pure protein
what is the oldest and least sensitive assay
Biuret assay
biuret assay is the oldest and least sensitive assay.
however…..
it is easy to do
Biuret assay is used to demonstrate……
the presence of peptide bonds
explain how the biuret assay demonstrates the presence of peptide bonds
in the presence of peptides, a copper (II) ion forms a violet colored coordination complex in an alkaline solution
the violet color produced has a maximum absorption at 550nm
what are interfering compounds in the biuret assay
glucose and sulfhydryl compounds
what does BCA Assay stand for
Bichinchonic Acid Assay
what assay is modified from the Biruet assay? explain it
BCA ASSAY
cuprous (+1) ion id generated from cuprous (+2) ion in the presence of protein in an alkaline environment
BCA reagent then chelates the cuprous ion and forms a BRIGHTLY COLORED COMPLEX with maximum absorbance at 562nm
does the BCA assay have good sensitivity? is it easy to use? any issues?
BCA assay has good sensitivity and is easy to use.
however, it is still susceptible to interfering agents such as:
REDUCING SUGARS (glucose) and metal chelators (EDTA)
ALSO, temperature and incubation times have a great influence on the variability of this assay
another name for the Coomassie Blue assay
Bradford Assay
the bradford (COOMASSIE BLUE) assay is based on…….
the shift of maximum absorbance of coomassie dye from 465nm to 595nm when it ineracts with proteins
another name for coomassie dye
Brilliant blue G250
How is the coomassie blue assay (bradford assay) able to accomplish its function?
the dye can bind nonpolar structures and cationic side chains (arginine and lysine) in acidic pH
true or false
the coomassie blue assay (bradford assay) expresses a linear relationship
TRUE
linear relationship between the shift in absorbance and the concentration of total protein
true or false
the bradford assay is insensitive, but simple to use
FALSE
it is sensitive and simple to use
what can interfere with the bradford assay?
high concentrations of detergent components (used for cell lysis purposes such as Triton X-100 and SDS)
which assay takes considerable more time than other methods?
Acid-digest Ninhydrin assay
explain how the acid-digest ninhydrin assay works
peptide bonds in proteins are broken using 6% sulfuric acid at 100 degrees celsius.
this breaks down the protein to its amino acids. the free emino end of the amino acids reacts with ninhydrin to form a DEEPER BLUE or PURPLE COLOR with maximum absorbance at 570nm
what are interfering compounds for the acid-digest Ninhydrin assay?
amino sugars and ammonium sulfate
