Foundation 1: MCAT

Enzyme

acts as a catalyst in biochemical reactions

Substrate

the molecules that serve as starting material for the reactions enzyme speed up 

Hormones

long distance chemical signals released by endocrine cells

Proteins

diverse and abundant molecules in living systems

Amylase

digestive enzyme, break down nutrients for absorption 

Lipase

digestive enzyme, break down nutrients for absorption 

Pepsin

digestive enzyme, break down nutrients for absorption 

Hemoglobulin

transport protein, carry substances through the blood or lymph

Actin

structure protein, build different structures 

Tubulin

structure protein, build different structures

Keratin

structure protein, build different structures 

Insulin

hormone signaling protein, coordinate the different body systems

Glucagon

hormone signaling protein, coordinate the different body systems

Antibodies

defense protein, defends body from foreign pathogens 

Myosin

contraction protein, carry out muscle contraction 

Albumin

Legume storage protein, provide the nutrients for early embryo development 

Amino Acids

monomers that make up proteins (polypeptides)

amino acids structure

alpha carbon bonded to COOH group (carboxyl), NH2 (amino), and H (hydrogen) at physiological pH, the amino group is protonated and bears a positive charge (NH3+) while the carboxyl group is deprotonated and bears a negative charge (COO-)

amino acid pKa>pH

protonated form

amino acid pKa<pH

deprotonated form

Protein Synthesis

DNA –transcription→ mRNA –translation→ protein

Polypeptides:

chain of amino acids held together by covalent bonds, known as peptide bonds 

Peptide bond

 formed in a dehydration synthesis reaction, the COOH reacts with the NH2 of another amino acid, releasing water and forming a bond, connect amino acids, rigid and planar bond that is stabilized by delocalization from the carbonyl oxygen and the nitrogen

amino acid orientation

the free amino group is on the left always and the free carboxyl group is on the right

n-terminus

free amino group

c-terminus

free carboxyl group

Primary structure

order of the amino acids

Secondary structure

interactions of the peptide backbone 

Alpha-helix

right hand coil of a single polypeptide chain, stabilized by hydrogen bonds every four carbons (3.6 amino acids per curl) 

Parallel Beta-Pleated sheet

a protein structure where the strands run in the same direction and are angled with less table hydrogen bonds

Anti-Parallel Beta-Pleated sheet

a protein structure where strands run in opposite directions, straight, and form stable hydrogen bonds between them 

Tertiary structure:

interactions of side chains

Hydrophobic effect:

The entropy driven clustering of non polar amino acid side chains in the protein due to the expelling of water from the structure to increase protein stability

Hydrogen Bond:

Polar interaction between side chains or separate polypeptides that can increase protein stability

Ionic Bonds

Salt bridges, strong interactions between negative and positive side chains 

Vander Waals Forces

Weak interactions between all molecules including amino acid

Disulfide Bonds

covalent linkages between the Sulphur containing side chains of cysteine that keep the polypeptides linked firmly together (favored in extracellular environments)

Quaternary structure

arrangement multiple polypeptide chain interactions

Conformational Entropy:

The protein folds and comes into its structure and decreases the entropy to offset other effects

Solvent Entropy

The hydrophobic effect, involving the water molecules being released from clustering structures creates a blockade from the solvent and the release increases the entropy of the water molecules

Molecular Chaperones:

Bind to fresh or partial polypeptides to prevent improper interactions

Chaperonins

large, cylindrical complexes that provide a proper environment for protein folding, after folding the protein is released

Denaturation

the alteration of protein structure, except the primary structure due to extreme conditions

Temperature Denaturation

higher temperatures increase the kinetic energy interfering with the non-covalent interactions that hold the protein together and expose interior hydrophobic regions

pH denaturation

increased pH can change the charge states of amino acids with acidic or basic side chains and interfere or break ionic and hydrogen bonds that hold the protein together

Chemical Agents denaturation

disrupt hydrogen bonds and unfold the proteins (urea)

Reversible Denaturation

A protein can refold into its functioning shape with the removal of the denaturing agent 

Irreversible Denaturation

Permanent damage to protein structure due to covalent bond interruption

Proper Conformation

correct primary, secondary, tertiary, and quaternary structure, if not misfolding can occur (active or folded form of a protein)

Conformation Stability

the various forces that act to keep a protein properly folded

Essential Amino Acids

Histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine cannot be synthesized from the body 

Non-Essential Amino Acid

Alanine, asparagine, aspartic acid, glutamic acid, serine, arginine, cysteine, glutamine, glycine, proline, tyrosine can be synthesized from the body

Phosphorylation

addition of a phosphate group, typically to serine, tyrosine, threonine, affects signaling pathways

Glycosylation

attachment of carbohydrate groups affecting protein folding, stability, and cell recognition 

Carbohydrate groups

monsaccharides, disaccharides, polysaccharides 

Acetylation and Methylation

epigenetic modifications that occur often on lysine, that influence gene expression and protein function

Epigenetic

heritable changes in gene expression that occur without alterations to the underlying DNA sequence

Histone Acetylation

acetyl groups are added to lysine residues on histone tails (protein part of chromatin), done by neutralization of the positive charge on the histones and weakening the interactions there is a relaxation of the chromatin allowing for the increased transcription factors

DNA Methylation

methyl groups are added to DNA, cytosine, then alter a transcription factor which activates or represses gene expression 

Histone Methylation

methyl groups are added to histone tails, lysine, and can alter gene expression 

Ubiquitination

attachment of ubiquitin to lysine residues, tagging them for protein degradation

Peptide Bond formation

nucleophilic addition-elimination reaction, lone pair on nitrogen in the amino group attacks the carbonyl hydrogen and attaches itself forming an amide bond, water leaves

Acid Hydrolysis

non-specific way of breaking a peptide bond, addition of heat allows the bond to break leaving multiple fragments of amino acids

Proteolysis

regulatory mechanism of gene expression, the specific breaking of a peptide bond , use of enzymes called proteases

Trypsin fragmentation

only fragments at the C terminus of Arginine and Lysine 

Pepsin fragmentation

prefers to fragment at the C terminus of phenylalanine, leucine, tyrosine, tryptophan

Elastase fragmentation

fragments at the C terminus of smaller amino acids

Thermolysin

fragments at the N-terminus of hydrophobic residues

Isoelectric Point:

a point on the pH scale in which an amino acid exists as neutral, no charge

pI: average of the pKas of all the functional groups in an amino acid

PI equation

pKa1 + pKa2 / 2

isoelectric focusing

found through a gel cascade path with and anode and cathode probing the proteins to move along the gradient as they need fit to neutralize the charges 

higher pI

more basic amino acids, needs a higher pH to balance 

the charges

lower pI

more acidic amino acids, needs lower pH to balance the charges 

Zwitterion:

a neutral molecule that contains both a positive charge and negative charge 

Protein Electrophoresis

separates protein based on their size and charge in a gel mixture that allows proteins to migrate toward the probe of the opposite charge

Native PAGE

proteins are separated by their native size and charge 

SDS PAGE

proteins are denatured and coated in a negative charge to be separated by their molecular weight

SDS

sodium dodecyl sulfate(reducing agent)

PAGE

poly acrylamide gel electrophoresis

SDS function

masks the proteins original charge and creates a linear chain to make an equal mass to charge ratio and separate them solely based on size

BME and DTT

reducing agents, break disulfide bonds

SDS-PAGE gel

stacking and resolving gels 

(low and high concentration) 

Buffer

maintain pH and conductivity

Tracking dye

visualize the separation (blue or silver stain)

Western Blotting:

gel electrophoresis that combines antibody interactions to identify a target protein with sensitivity and specificity 

SDS page gel

protein separation

WB Primary Antibody

Targets the protein

WB Secondary Antibody

Conjugates the enzyme that binds to the protein

WB Materials

SDS page gel

Transfer Membrane 

Primary Antibody 

Secondary Antibody

Buffer

Applications of WB

Quantifying protein expression, analyzing post-translational modifications, and protein identification

Applications of SDSPAGE

 identify proteins, analyze their purity, and determine the molecular weight

Electroblotting

transfers protein samples from electrophoresis gel to a membrane using an electric field

Enzyme-linked Immunoassay (ELIZA):

uses antibodies to detect and quantify directly in a liquid sample, a protein binds to a solid surface and a signal if produced by an enzyme-linked antibody often color change 

Southern Blotting

Used to detect specific DNA sequences from a complex sample

SB Process

gel electrophoresis separates the DNA fragments, then transferred to a membrane then   incubated, where a labeled DNA probe will hybridize the fragments for detection 

Applications of SB

Testing for genetic disorders, analyzing gene structure/ organization, detecting specific sequences 

Northern Blotting:

Used to detect specific RNA sequences from a complex sample 

NB Process

extracted RNA samples are separated in denaturing conditions by gel electrophoresis, and transferred to an incubated membrane, then probed for detection

Mobile Phase:

Solvent moving through the column

Stationary Phase:

Substances that stay fixed inside the column