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Biomedical Lab III > Lab 4 > Flashcards

Lab 4 Flashcards

1
Q

Define Enzymes

A

enzymes are proteins that act as catalysts in specific biochemical reactions, including the metabolism of drugs

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2
Q

explain the fate of drugs that are metabolized

A

they have undergone a chemical change that will dictate their pharmacological and toxicological profiles

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3
Q

true or false

enzymes involved in drug metabolism also metabolize endogenous substances such as hormones, vitamins, and lipids

A

TRUE

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4
Q

_____ is the primary site of metabolism for most drugs

A

the liver

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5
Q

as mentioned, the liver is the primary site of metabolism for most drugs.

name other significant organs for drug metabolism

A

kidneys
lungs
GI tract
blood
nervous tissue
skin

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6
Q

define first pass metabolism

A

the ability of the liver (and extrahepatic tissues) to metabolize drugs before they reach systemic blood circulation

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7
Q

where are cytochrome P450 enzymes found?

A

in liver cells in the membrane of the endoplasmic reticulum

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8
Q

name 2 examples of endogenous molecules

A

nutrients and hormones

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9
Q

name 2 examples of exogenous molecules

A

drugs and xenobiotics

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10
Q

the metabolism of both endogenous and exogenous molecules are classified as…..

A

phase 1 and phase 2

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11
Q

do phase 1 and phase 2 metabolism occur in most tissues?

A

yes (liver, kidney, skin, and lungs)

but the LIVER is the PRINCIPAL SITE for DRUG METABOLISM

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12
Q

the primary purpose of liver metabolism is __________

A

drug inactivation aka detoxification

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13
Q

the primary purpose of liver metabolism is drug inactivation (detoxification)

what is a concern with this

A

some drug metabolites ARE pharmacologically active and sometimes may even be more potent than the parent compound

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14
Q

Prodrugs are designed for….

A

conversion to an active moiety in the liver

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15
Q

what can you say about molecules that are very lipophilic?

A

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

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16
Q

TRUE OR FALSE

drugs always undergo phase 1 and phase 2 of metabolism

A

FALSE

they may only undergo phase 1 or 2 depending on their functional groups and polarity

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17
Q

what does the S9 fraction contain?

A

both phase 1 and phase 2 drug-metabolizing enzymes

phase 1 = cytochrome P450 in the microsomes

phase 2 = cytosolic fraction

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18
Q

_______ is a valuable tool to study the in vitro metabolism of drugs.

explain

A

S9 fraction bc it contains both phase 1 (cytochrome P450 in the microsomes) and phase 2 (cytosolic enzymes) drug metabolizing enzymes

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19
Q

how can you predict the metabolic fate of the compound of interest? (drug)

A

by incubating it with the S9 fraction

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20
Q

the quality of the S9 fraction and other enzyme extracts is generally based on……..

A

the enzymatic activity present in 1mg of protein

when COMPARING enzyme extracts, the concentration of the protein in the extract must also be analyzed

21
Q

true or false

there are few types of assays one can use to determine the protein concentration of a sample

A

FALSE – there are many types

22
Q

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?

A

-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

23
Q

How do you determine the concentration of an unknown sample?

A

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

24
Q

“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?

A

interpolation

25
Q

what can you sometimes do to ensure that the absorbance of the unknown falls within the standard curve absorbances

A

dilute your protein sample

26
Q

what is extrapolation

A

calculating for protein values outside of the standard curve values

27
Q

is there anything wrong with doing extrapolation?

A

this can result in error in calculating the protein concentration

28
Q

explain how standard curves are usually plotted

A

Y-axis = absorbance
X-axis = protein concentration of the standards

29
Q

what protein should be used for the standard curve?

why?

A

BSA (bovine serum albumin)

this is because it is a well-defined, pure protein

30
Q

what is the oldest and least sensitive assay

A

Biuret assay

31
Q

biuret assay is the oldest and least sensitive assay.

however…..

A

it is easy to do

32
Q

Biuret assay is used to demonstrate……

A

the presence of peptide bonds

33
Q

explain how the biuret assay demonstrates the presence of peptide bonds

A

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

34
Q

what are interfering compounds in the biuret assay

A

glucose and sulfhydryl compounds

35
Q

what does BCA Assay stand for

A

Bichinchonic Acid Assay

36
Q

what assay is modified from the Biruet assay? explain it

A

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

37
Q

does the BCA assay have good sensitivity? is it easy to use? any issues?

A

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

38
Q

another name for the Coomassie Blue assay

A

Bradford Assay

39
Q

the bradford (COOMASSIE BLUE) assay is based on…….

A

the shift of maximum absorbance of coomassie dye from 465nm to 595nm when it ineracts with proteins

40
Q

another name for coomassie dye

A

Brilliant blue G250

41
Q

How is the coomassie blue assay (bradford assay) able to accomplish its function?

A

the dye can bind nonpolar structures and cationic side chains (arginine and lysine) in acidic pH

42
Q

true or false

the coomassie blue assay (bradford assay) expresses a linear relationship

A

TRUE

linear relationship between the shift in absorbance and the concentration of total protein

43
Q

true or false

the bradford assay is insensitive, but simple to use

A

FALSE

it is sensitive and simple to use

44
Q

what can interfere with the bradford assay?

A

high concentrations of detergent components (used for cell lysis purposes such as Triton X-100 and SDS)

45
Q

which assay takes considerable more time than other methods?

A

Acid-digest Ninhydrin assay

46
Q

explain how the acid-digest ninhydrin assay works

A

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

47
Q

what are interfering compounds for the acid-digest Ninhydrin assay?

A

amino sugars and ammonium sulfate

48
Q
A

Biomedical Lab III (10 decks)

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