Biochem Flashcards
Glycine
Gly, G, nonpolar
Alanine
Ala, A, nonpolar
Valine
Val, V, nonpolar
Leucine
Leu, L, nonpolar
Isoleucine
Ile, I, nonpolar
Phenylalanine
Phe, F, nonpolar
Tryptophan
Trp, W, nonpolar
Methionine
Met, M, Nonpolar
Proline
Pro, P, nonpolar
Serine
Ser, S (Polar, Neutral)
Threonine
Thr, T (Polar, Neutral)
Tyrosine
Tyr, Y (Polar, Neutral)
Cysteine
Cys, C (Polar, Neutral)
Asparagine
Asn, N (Polar, Neutral)
Glutamine
Gln, Q (Polar, Neutral)
Aspertate
Asp, D (negative)
Glutamate
Glu, E, negatively charged
Lysine
Lys, K, positive
Arginine
Arg, R, positive
Histidine
His, H, positive
Characteristics of alpha strands
*i and i+4
* all the side chains are projected on the outside
* NH bonds with CO on top
Beta Strand Characteristics
*fully extended polypeptide chain
* multiple B strands from B sheet
* parallel and anti parallel
Parallel and anti parallel
Parallel - H bond by NH and 2 strands from opp amino acids (turn to my classmate next to me but ignore her and hold out hands to the girl in front of her and person behind her
anti parallel - shake hands with the girl right next to her
Loops/ Turns characteristics
* i and i+3
Glycine and proline are the most common
What happens when not glycine in hole of collagen
Osteogenesis - bc bigger structures will push away other molecules and nothing will become tightly packed.
Characteristics of tertiary structure
Polar chains point out to face aqueous solution, non polar buries to hide from h20
Alpha keratin characteristics
* only helices - that wrap around each other
* held together by van der waals, salt bridges and disulfide bonds
*interactions at the every 7 aminos
Characteristics of collagen
* bones and cartilages
* 3 helices wrapped around each other
*glycine is every 3rd amino
Can proteins be refolded, why or why not?
It can fold but it is not guaranteed that it will return to the same structure that it was - and will most likely be an inert protein
Urea denatures what
2nd structures (alpha, beta, turns)
Van der waals, H bonding, salt bridges
guanidinium chloride denatures what
2nd structures (alpha, beta, turns)
Beta Mercator ethanol denatures what and how
Is a reducing agent that breaks covalent bonds (S-S)
Process of harvesting a protein
1) break membrane to bring proteins outside of cell
- can break membrane by lysing, sqessing the cell mechanically (homogenized pressure via air, sonicator - break via sound waves, break chemically
2) place in centrifuge to separate soluble proteins from insoluble cell debris (plasma membrane)
What is the ion exchange column chromatography?
What is the process?
* cation and anion exchange
1) choose a buffer that will attract the protein of interest
2) elution from column- flush with high concentration of similarly charged ion to replace the protein of interest and it can flush out
What charge product would you get with cation and anion exchange?
Anion - negative charge ( which means the charge on the column is +)
Cation - positiv charge (column is -)
What is size exclusion/ gel filtration chromatography
* separate protein based on size
- basically the larger proteins reach the end the fastes bc its not help up by the beads (too big to go in) and the smaller the beads are…the slower to reach
Affinity chromatography
Load the column with a molecule/thing that only the protein of interest would be attracted to
Nickel affinity chromatography
1) add a sequence of codes that will translate (make) 6 Histidines at the beginning or end of the sequence. The nitrogen on histidine is the ONLY one that will form a coordination bond with the Nickel - and because there are 6 histidines on this protein… it would have a higher affinity to the nickel than any other protein ( because the histidine is exposed etc)
2) flush with imidazole bc its structure is very similar to histidine and it will replace the histidine and the B protein with His6 will flush down
Salting out
The higher concentration of salt ammonium sulfate (NH4SO4) that you add - the more that it will get in between the protein and water. It will bond with the water and the protein will precipitate out (bc its not soluble any more)
What is SDS?
1) detergent that denatures the protein completely
2) add lots of sulfate to make the protein charge very negative
3) will break all interactions except S-S bonds (covalent)
SDS - Page (how do the charges move)
1) add a positive charge at the end of the terminal so that the negatively charged proteins can migrate towards it . The proteins are separated by size - larger molecules are slower to migrate, smaller ones migrate faster.
BME what test is it found in?
SDS Page
It is a reducing agent that breaks S-S bonds
Tracking dye
SDS Page - stains blue to see how far things traveled
Glycerol
SDS page
Makes the solution heavy so the things stay in the wells and migrate down
What happens if you load something that is a dimer connected by disulfide bond
- in a reducing gel (with BME)
- in a non reducing gel (no BME)
* there would be 2 lines (right on top of each other) with the size of the monomer)
there would be one line with the monomer size x2
What is Page
Gel/ mesh network
What is Isoelectric Focusing
* Gel electrophoresis with PI
What is 2D gel
Isoelectric focusing with SDS Page
* isoelectric -PI: Pi, left vs right
* SDS page: size, top vs bottom
Define antigen
Foreign invaders
Define antibody
A protein produced by the body to defend
Define epitope
Recognition site that antibodies attach to
Define components of antibodies (what is it made up of)
Look at notes
What is monoclonal antibodies and why is it beneficial
It is produced by one B cell, so you know what exact properties that you’re getting
What is western blot and describe the process
SDS page and antibodies combined and used for detection
What is ELISA
An INDIRECT diagnostic test - look for antibodies by monitoring the response of the body
What is Sandwich ELISA
A direct test which tests for antigens ex. COVID test
What is Chromatogram
Uses gel filtration (size exclusion)
What are the 2 models of specificity
Lock and key
Induced fit
Define initial velocity
Velocity at the early stage of reaction
Define steady state kinetics
Rather of formation of ES complex = rate of dissociation of ES complex and therefore the concentration of ES complex remains steady
Equation of rate of formation
ES = k1 [E][S]
Rate of dissociation of ES complex
(K-1, K2)ES or K-1 (ES) & K2 (ES)
What is Et
Et= E + ES
Define theoretical max velocity
Max velocity is reached when all free enzymes exist as ES complex
Define the relationship of Km and velocity
Km is the substrate concentration when the initial velocity equals Vmax/2
*km is the substrate concencetration required to reach half of Vmax
What is the equation of y-int
Y-int= 1/Vmax & Vm= 1/ y-int
What is the equation of slope
Slope= Km/Vmax & Km= slope x Vmax
What is the Michaeli - Menton Equation
Vo = Vmax [S]/ [S] + Km
Characteristics of Km
* The lower Km, the higher the affinity an enzyme has to substrate
Define Activity
It is the rate of reaction, amount of product formed over time
Define specific activity
It is the amount of product formed by 1 mg of the enzyme
Define Turnover number
It is the amount of times an enzyme turns over, when it is operating in its max velocity
What are the characteristics of Turnover
Higher the turnover, the faster and more product is being produced
Equation for Kcat and the characteristics of it
Kcat=Vmax/[Et]
Alanine, Ala, A, Nonpolar
Glycine, Gly, G, Non polar
Valine, Val, V, Nonpolar
Leucine, Leu, L, Nonpolar
Isoleucine, Ile, I, Nonpolar
Structure
Phenylalanine, Phe, F, Nonpolar
Tryptophan, Trp, W, Nonpolar
Methionine, Met, M, Nonpolar
Proline, Pro, P, Nonpolar
Serine, Ser, S, Polar neutral
Threonine, Thr, T, Nonpolar
Tyrosine, Tyr, Y, Polar neutral
Asparagine, Asp, N, Polar neutral
Cysteine, Cys, C, polar netural
Glutamine, Gln, Q, Polar neutral
Aspertate, Asp, D, negative
Glutamate, Glu, E, negative
Lysine, Lys, K, positive
Histidine, His, H, positive
Arginine, Arg, R, Positive
Competitive
Uncompetitive
Non competitive
What happens to Vmax, Km, and Y-int with competitive, NC, and UC
What is the y=mx +b for Vmax etc,
what is the formula for Vmax
what is the formula for km
what is the formula for x-int
What are the 2 types of bisubstrate reaction?
Sequentia and Ping Pong
What is sequential?
It can be ordered or random
What is ping pong
What is the free energy change equation for reaction
What to do if Et is given in mass
Convert mass to moles using the MW of the protein
What are the characteristics of Km and Kcat?
Higher Kcat is better
lower km is better
Velocity of reaction when [S] = 10% of km
Acids catalysts make…..
Better leaving groups and it increases the electrophicity of the carbonyl (electrophile)
Basic catalyst makes….
Better nuclephiles by increasing the nucleophility of the nucleophile
What is the mode of recognition for cymotripsin
What is the mode of recognition for trypsin
What is the mode of recognition for elastase
Small side chains
What is the mechanism of Chymotrypsin
What are the modes of recognition for chymotripsin, trypsin, and elastase
What is the mechanism for cysteine protease
What is the mechanism for aspartyl protease
Why are amide bonds stable in protease reaction
Amide bonds are very unreactive towards any nucleophile
What is the role of the catalytic triad
Bodies way of providing basic and acidic conditions
* Serine forms the catalytic bond
* Histidine provides the proton to make serine more nucleophilic
*Aspertates COO- makes the positive charge on Histidine more stable
Site Directed Mutagenesis
Process of Elisa Sandwich
Describe the process of ELISA
What is the equation that shows the process from enzyme to product
What are the reaction rates of the equations
In the M. M equation, which are constants and which are variables
Equation for Vo in terms of Km
Equation for Kcat and the characteristics of it
Kcat = Vmax/ Et
Carbon dioxide combines with water to make what?
Carbonic acid –> which disassociates into bicarbonate acid
What is the mechanism from CO2 and H20 –> carbonic acid
How can you make the mechanism of making carbonic acid faster?
By making water more acidic (and this is done by Zn)
What is the goal of metalloprotease and what is the mechanism?
Explain this graph
What is regulation?
Factors that helps the enzyme function at the right place and time.
What are the 5 ways that enzymes are regulated
- ATCase
- Proteolytic cleavage
- Multiple forms of the same enzyme
- Control by covalent modification (phosphate hat makes enzyme active)
- Control by gene expression
