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AA and Proteins
Robert F. Waters, PhD
Overview

Proteins
– Structural
– Enzymatic
Amino Acids
 Henderson-Hasselbach Equation
 Acidity and Alkalinity
 Gas exchange
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Proteins
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Polypeptides with peptide bonds
– Peptide bonds
» Endergonic (Consume energy)

Need energy and do not occur spontaneously
Structural proteins
 Soluble proteins (Enzymes)
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Protein Structure
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Primary protein structure
– Sequence of amino acids
» Nomenclature: ala-glu-gly (N-terminus to C-terminus)
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alanylglutamylglycine
Secondary structure
– -helix and -sheet
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Tertiary structure
– 3-dimensional folding
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Quaternary structure
– Multiple subunits of tertiary structures
Protein Structure: Primary
Amino terminus
Carboxyl terminus
Protein Structure:Secondary
Protein Structure: Tertiary and
Quaternary
Forces That Stabilize Proteins

Ionic bond
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Hydrogen bonding
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Hydrophobic interactions
– Hydrocarbons in aqueous solution have force
association with adjacent hydrocarbons by
rearrangement of surrounding water molecules
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Van der Waals interactions
– Weak electrostatic attractions
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Denaturation of Proteins
Soluble Proteins Precipitate
Dehydration
Heat
Radiation
pH
Cold
Pressure
Chemicals
Excessive vibrational energy (Microwaves)
Natural organic substances (e.g., urea)
Reducing agents
– e.g., Mercaptoethanol HS-CH2-CH2-OH
» Blocks disulfide bond formation
Post-Translational Denaturation

Associated with Golgi Apparatus
– Packaging
– Folding
Herbicides
 Pesticides
 Neurotoxins (snake venom)
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Example of Precipitation by
Acidification
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Milk proteins (Two Main Types)
– Casein
» 1-casein, s2-casein, -casein, -casein, -lactalbumin,
-lactoglobulin
– Whey (serum protein)
» Serum albumen, immunoglobulins, lactoferrin
Casein separated from whey by acidification to casein pI of 6.0.
– Like adding citrus to coffee with cream
» Serum (whey)proteins remain in solution while casein
precipitates
– Casein with lipids forms micelles (opaqueness of milk)
Whey protein (hydrophilic) is used as protein addition to drinks,
thickeners
Casein is an excellent emulsifier in the addition of flavoring agents
Vitamins and Minerals May Give
Color to Protein
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When a vitamin or mineral gives a protein color is called a
chromophore
FAD or FMN added to apoproteins to form flavoproteins
give a yellowish color
Iron with myoglobin in meat
– Ranges in color from brown to bright red
– White poultry meat has low myoglobin
– Dark meat has high myoglobin content
– Veal and pork have less myoglobin than beef
Myoglobin and hemoglobin without iron are colorless
Myoglobin and hemoglobin with iron are pink to red
Cooking meat dissociates heme to protein, iron and other
complexes and produces brown to tan color
Quantifying Protein in Solution

Based on absorption spectra of aromatic amino acids (~280nm)
– Tryptophan, tyrosine, phenylalanine
– Different proteins may vary in aromatic amino acids but absorption
spectra variation is still useful
Zymogen System
Series of enzyme activations for the
digestion of protein into amino acids
 Protection mechanism against autolysis of
endogenous proteins
 Begins mainly in the stomach and proceeds
to intestine
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The Stomach:Overview
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Stomach not a very absorptive organ but—
– Water, ETOH, short and medium chain FAs are
absorbed
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Gastric mucosa
– Chief cells, parietal cells and mucous cells
» Produce gastric juices called gastrins
Summary of Stomach Gastrins
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Parietal Cells of Stomach
– Secrete HCL
» Denaturation, very little digestion
– Secrete Vitamin B12 intrinsic factor
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Chief Cells of Stomach
– Secrete gastric lipase
– Secrete pepsinogens
» Activated to pepsin by HCL
» Activated to pepsin by sutolysis
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Pepsin cleaves proteins into large oligopeptides (peptones)
Mucosa cells
– Secrete bicarbonate and mucus
Stomach Summary Cont:
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Stimulation of gastric secretions (Gastrin)
–
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–
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Protein itself
Vagal stimulation
Calcium ions
Alkalination of the stomach
Gastrin stimulates –
– HCL production (parietal cells)
» HCL inhibits gastrin production
» NaCl necessary for HCL production
– Mucin (mucous cells)
– Pepsinogen production (chief cells)
» Activated by HCL to Pepsin
Zymogens
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Proenzymes (zymogens) packaged as
zymogen granules in pancreas
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Pancreatic zymogens are serine proteases
– Trypsin (less than proenzyme form)
– Chymotrypsin
– Elastase
Enteropeptidase Activation
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Enteropeptidase produced in intestinal
brush border
– Activates trypsin from trypsinogen
» Some activation of trypsin by autolysis
» Trypsin activates chymotrypsinogen, elastase and
carboxypeptidase A and B (possibly some
aminopeptidases)
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Carboxypeptidases and Aminopeptidases
are called Exopeptidases
Graphic RepresentationEnteropeptidases and Cascading
Phosphorous Containing Nerve
Gases
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Initial studies with acetylcholine esterase
Nerve gas DFP
– Diisopropylfluorophosphate
– Attacks serine hydroxyl groups in enzymes like
acteylcholine esterase AND serine proteases like
chymotrypsin
» Attacks serine 195 in chymotrypsin
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DFP acts as a pseudo-substrate for the enzymes
DFP stops enzymatic reaction
Graphical Representation of DFP
Cysteine Proteases
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Attack sulfhydryl group on cysteine in protein
Examples (mammals have similar proteases)
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Papain (papaya)
Bromelain (pineapple)
Ficin (fig)
Actinidin (kiwi fruit)
Caricain, chymopapain, glycyl endopeptidase (from
latex portion of papaya tree)
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Lysosomal Proteases
Active at lower lysosomal pH
Cathepsins
– Cathepsin B (Most abundant)
» Endopeptidase and Exopeptidase
– Cathepsin H (Aminopeptidase)
– Cathepsin K (Abundant in bone resorbing osteoclasts
» Absence causes fragile small bones
– Cathepsin C (dipeptidyl peptidase)
» Removes N-terminus dipeptides activating intracellular
proteins and maybe other Cathepsins
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Bleomycin hydrolase
– Bleomycin is an anti-cancer drug
– Bleomycin hydrolase breaks down bleomycin
» Unfortunately cancer cells have high amounts of this enzyme
causing drug resistance
– Papain-like activity that also binds to DNA?
Cysteinyl Aspartate-Specific
Proteases (Caspases)
Involved in programmed cell death
(Apoptosis)
 Activation of Interleukins
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– Caspase-1 (AKA: Interleukin-1-converting
Enzyme or ICE)
» Cleaves pro-interleukin-1 to form the active
interleukin-1
Aspartate Proteases (Pepsin-Like)
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Example is gastric proteinase – Gastricsin
– Has similar activity as rennin (chymosin) from
the fourth stomach of calf
» Causes rapid clotting of milk
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Used in cheese manufacturing
– Serum protein Renin (NOT rennin) is similar as
well
Protease Inhibitors-Exogenous
Leupeptin (Inhibits trypsin)
 Boronic Acids (Inhibit serine proteases)
 Pepstatin (Inhibit aspartic proteases)
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– Were “Lead Compounds” for the formation of
HIV protease inhibitors
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Mercaptans (Inhibit Zn++ metalloproteases)
– Bind to Zn++ in some metalloproteases
» Captopril
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Drug that inhibits Angiotensin-Converting-Enzyme (ACE)
Endogenous Protease Inhibitors
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Trypsin activation in pancreas would be disastrous
– Pancreatic Trypsin Inhibitor
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Serpins (Blood)
– Inhibit serine proteases
– 10% of the total protein in blood
» 1-protease Inhibitor (1-antitrypsin)
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Found in -globulin fraction of blood
» NOTE:One form of emphysema is the hereditary absence of
1-antitrypsin
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Without this inhibitor, tissue will degrade excessively, e.g.
elastin, collagen and proteoglycans
Protease Activities-Serine
Proteases
Amino Acids
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Last one described was threonine in 1938
Stereospecific (L-Configuration exclusively)
– D-Amino acids found in bacteria
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Chemical properties associated with
stereospecificity and side groups
Easily ionized in aqueous solution
Produces a zwitterion (dipolar chemical structure
with + and – charges
Zwitterion effect causes crystalline form of amino
acids to have high decomposition temperatures
above 200o centigrade
– Similar to electrostatic forces holding and NaCl lattice
together
Overall Structure of Amino
Acids
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-Carbon, Carboxyl Group, Amino Group
– Except imino amino acids
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Enantiomeres (L-Amino acids in proteins)
– D (Dextro) and L (laevo)
Amino Acid Structures-Neutral
Side Groups
Amino Acid Structures-Aromatic
and Acidic Side Chains
Amino Acid Structures-Positive
Side Groups
Amino Acid Structures-Polar
Side Groups
Classification by Polarity
Essential Amino Acids
PVT TIM HALL
 Phenylalanine, valine, trptophan, threonine,
isoleucine, methionine, histidine, arginine
(neonate-child), leucine, lysine
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Non-Essential Amino Acids
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Synthesized by humans
– Serine, glycine, cysteine, alanine, aspartate,
asparagine, glutamate, glutamine, proline,
arginine (adult), tyrosine (from phenylalanine)
Non-Protein Amino Acids
Citrulline is a product of L-arginine
synthesis (urea cycle) and NO (Nitric
Oxide) metabolism
 Creatinine is derived from muscle
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– Plasma amounts  to muscle mass
Ornithine, taurine, homocysteine
 Biogenic Amine Compounds
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– Dopamine, serotonin, histamine
Amino Acids and pH
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pH = -log10 [H+] ion concentration
– Alkalinity vs. acidity
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Absorption of AA and pH
Henderson-Hasselbach Equation
(H-H)
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HA is protonated form
– Conjugate acid or associated form
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A- is unprotonated form
– Conjugate base or dissociated form
Protonation occurs in acidic solutions
 Removal of protons in more alkaline
solutions
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H-H Continued:
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Acidic amino acids
– Glutamate and aspartate are negatively charged
acidic amino acids at physiological pH
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Basic amino acids
– Arginine, lysine, and histidine are positively
charged amino acids at physiological pH
pK Values for Amino Acids
Titration of Amino Acids with
(OH ) NaOH-Glycine
Titration of Amino Acids with
(OH ) NaOH-Histidine
Aspects of Titration
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pI = Isoelectric Point
– pH where net charges equal zero (0)
» Histidine pI = 7.7 (6.0 + 9.3)/2 = 7.7
» pHm = (1.8 + 6.0)/2 = 3.9
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pHm = Maximum Charge
– pH where number of positive and negative charges are
maximal
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Buffering Range
– Range where change in pH is minimal
– Approximately +1 pH above pK to –1 pH below pK
value
» e.g. if a pK value = 6.0, then the buffering range around this
pK would be 5.0 – 7.0
Importance of Regulating pH
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Denaturation of Protein
– Enzymes
» Charge distribution
» Hydrolysis of bonds
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Charge changes on amino acids
– Substrate specificity
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Hydroxyl amino acids
– Serine, threonine, tyrosine
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Quaternary structure
– Proteins, hemoglobin binding of oxygen
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pK of amino group
– Free amino acid amino group pK = 9.5
– Amino group in a polypeptide pK = 8.0
Extremes of pH
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Acidosis to Alkalosis
– Acidotic condition pH=7.0
– Alkalotic condition pH=8.0
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Normal pH = 7.4
– Venous blood pH = 7.35
– Arterial blood pH = 7.45
» Higher altitude arterial pH = 7.49
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Most extreme limits are pH = 7.0 – 8.0
Acidosis
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Acidosis
– Hyperventilation
» Controlled by mid brain
– Acidotic coma
» Reduction in myocardial contractions
» Reduction in catacholamines (e.g., histamine)
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Reduce vascular tone (shock)
Pumping blood against a non-resistant wall
» Hypooxygenated (Cannot bring in enough O2)
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Excess protons block hemoglobin binding of O2
– “Bohr Effect”
Acidosis Continued:
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Sources of Protons
– Volatile acids (Respiratory)
– First conversion is carbonic anhydrase
– Second reaction is spontaneous

3
CO2  H 2O  H 2CO3  HCO  H
– Non-volatile acids
» Lactate (Metabolic)
» Ketones (Liver produces these thinking there is a lack of
glucose)
» Sulfuric (From Cysteine Degradation)

Alkalosis
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Alkalosis
– Hypoventilation
– Tetany (Sustained uncontrolled muscle
contraction)
» Muscle contraction controlled mainly by Na in
neuron and Ca in muscle

2
H
P
O

HPO
» Related to phosphate
2
4
4
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Monobasic and dibasic forms
Under alkalosis with the addition of [OH] you form water
Also drive reaction to the right

H 2 P O4  HPO42  H   OH 
 H
Alkalosis Continued
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Dibasic form of phosphate can chelate
soluble calcium better than monobasic form
to form calcium phosphate

2
4
H 2 P O4  HPO  H
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Does not change blood concentration of
calcium only amount that is soluble
Alkalosis Continued
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What does calcium do in nerve impulses?
– Inhibits transport of sodium into nerve during
the process of depolarization due to a nerve
impulse
– Under alkalotic conditions more calcium is
chelated and removes the controlled blockage
of sodium into nerve (uncontrolled influx of
sodium into nerve)
» More nerve depolarization
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Causes the sustained uncontrolled muscle contraction
during alkalosis (tetany)
Air Exchange
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Partial Pressures of Gases in Air
– 80% N2
– 20% O2
– .03% CO2
1 atm = 760mm Hg (Sea level)
 Example:
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– pN2 = .79 * 760 = 608mm Hg
– pO2 = .20 * 760 = 152mm Hg
– pCO2 = .0003 * 760 = 0.23mm Hg
Gas Exchange
pCO2 of blood is around 40mm Hg
 pCO2 in lungs is around 35mm Hg
 Partial pressures of respiring cells higher
yet
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