10.

Amino acids/Proteins
Chapter 17
 Protein - More than an Energy Source

Proteins / polypeptides - chains formed by

the condensation/combination of 20 different
 - amino acids.

• Polypeptides - may be di-, tri -, etc; up to 10 a.a.

• Proteins - longer than 10 a.a. units; ie. MW>10,000
 Amino Acids - Protein building blocks

An amino acid is a compound having both a carboxyl

group(-COOH) and an amino group(-NH2).

H All amino acids from protein

have the -NH2 attached at
H 2N C COOH
the C  to the –COOH
R (as well as the H- & R-).

All naturally occurring -amino acids, except

glycine (R=H), are chiral and the ‘L’ stereoisomer.
 H
H 2N C COOH
There are 20 -amino acids
R in naturally occurring protein.
By convention the -NH2 is
placed ‘to the left’.

Each aa has a ‘common’ name often ending in ‘-ine’.

There are ~150 other physiologically important

amino acids, GABA (a neurotransmitter).
Amino Acids - 1
Amino Acids - 2
 Amino acids
• Contain both an acidic functional group
(COOH) and a basic one (-NH2), NH or N
• Thus reactions are highly pH dependent
 pH dependent properties
• Zwitterionic structures contain both N-H+
and COO-.
• At low pH, protonate COO-.
• At higher pH : lose H on N
• Isoelectric pH: differs for each amino acid
(due to structural differences)
 Leucine ionic forms
• Cation below pH 2.4
• Neutral between pH 2.4 and 9.6
• Anionic above pH 9.6
 Leucine zwitterion
• pH>2.4
• pH < 9.6
 Peptides – Buildup/Breakdown
O O

H dehydration
H2N CH C OH + H N CH C OH
hydrolysis

CH3 H
alanine (ala) glycine (gly)

Peptide or amide linkage

O O
H
H2N CH C N CH C OH + H2 O

CH3 H carboxylic acid

amine
end
end
alanylglycine (ala-gly) -a dipeptide
 Dipeptides
• Consider the 2 amino acids glycine (G) and
alanine (A).
• How many dipeptides can be made if these
are randomly mixed?
• GG, AA, GA and AG
• N terminal on LHS; C terminal on RHS
 Tripeptides
• Consider amino acids Glycine (G), Alanine
(A) and Phenylalanine (P)
• How many different tripeptides are possible
if each amino acid must be present?
 Possible tripeptides
• 3 choices for the N-terminal amino acid
• 2 choices for middle
• 1 choice for the C terminal amino acid
• Thus 3 x2 x1 =6 choices if each aa must be
present.
• But total number possible is 3 x3x3 =27;
includes AAA, PPP, GGG etc
 Protein Structure
• The only unambiguous way to determine
the overall structure of any molecule
is…………..

• Sequence of amino acids can be determined

using the enzyme carboxypeptidase (cleaves
one aa at a time from the C terminal end)
 Levels of Protein Structure
Primary structure - the sequence of amino acids
in the peptide chain and the location of the disulfide
bridges.
Secondary structure - a description of the
conformation/ shape of the backbone of the protein.
Tertiary structure - a description of the 3D
structure of the entire polypeptide.

If the protein has more than one chain it can have a

quaternary structure.
 Some Protein Sequences

Phe - Gln Oxytocin – contracts smooth muscle

(induces ‘labour’)
Tyr Asn
Cys Cys - Pro - Arg - Gly
S-S

Ile - Gln
Vasopressin - diuretic
Tyr Asn
Cys Cys - Pro - Leu - Gly
S-S
 Val- Ile-Gly Insulin (21 + 30)
Glu
Gln
Cys Cys-Ser-Leu-Tyr-Gln-Leu-Glu-Asn-Tyr-Cys-Asn
Cys-Thr-Ser-Ile

Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly
Leu Glu

His Arg

Gln- Asn-Val-Phe Gly

Thr-Lys-Pro-Thr-Tyr-Phe- Phe
 Secondary structure of Proteins
• Is the fixed arrangement of amino acids
resulting from interactions between amide
linkages that are close to each other in the
protein chain
• Interactions can be hydrogen bonds (~ 5
kcal/mol each)
• Many H bonds are sufficient to define the
shape
 Ionic Interactions in Proteins
• “salt bridges”
• Involve COO- and remote NH3+ groups
• Along with H bonding and dispersion
forces, these are responsible for the overall
shape or “conformation” of the protein
Secondary (20) Structure - sheets
O R H
N
N
H O
H – bond
O H
N
N
H R O

sheets/strands, eg. fingernails, silk

Secondary Structure(20) - the -Helix

H-bonding -
intramolecular
 Tertiary Structure of Proteins
• Arises from weaker attractive forces (non
polar dispersion forces) between
hydrophobic parts of the same chain that are
widely separated in the primary structure,
but close in space
• “intramolecular”
• Results in chain twisting and folding
 Dispersion forces
• Attractive when nuclei are separated by the
sum of their van der Waals radii
 Tertiary structure of protein:
braids and globs
• Collagen-a fibrous protein (precursor of
gelatin) has a triple helix structure-some
elasticity due to interchain interactions
• Hemoglobin (a globular protein)
Tertiary Structure (30) - braids & globs

collagen

hemoglobin
Hemoglobin(H) and Myoglobin (M)
• H has 4 polypeptide chains : carries O2, CO2
and H+ in the blood, and possesses
quaternary structure
• M has a single chain of 153 amino acids:
carries O2 from the blood vessels to the
muscles and stores it until needed.
• Both have Fe II containing heme unit in
each chain that binds O2.
Myoglobin Structure
 To summarize
• Myoglobin cannot have quaternary structure
since it has only one polypeptide chain
• Hemoglobin has 4 polypeptide chains and
possesses quaternary structure
 Enzyme structure
• Many enzymes are proteins and their
specific binding properties to a substrate
depend on their overall molecular shape or
“conformation”
Lock and key mechanism for activity
Active Site of Enzymes
Denaturation -
any physical or chemical process that changes
the protein structure and makes it incapable of
performing its normal function.
Whether denaturation is reversible depends
on the protein and the extent of denaturation.
Examples:
 heating egg whites (irreversible)
 ‘permanent’ waving of hair (reversible)
 Protein Chemistry and your hair
• Forces combining to keep hair (a) straight
(b) in loose waves or (c) in tight curls are:

• Disulfide linkages (part of 10 structure)

• Salt bridges
• Hydrogen bonds
 Protein in Human hair
• Keratin (fibrous protein) has the S
containing amino acid cystine (14~18%) .
• S-S bonds (disulphide linkages) between
cystine units give hair its strength by
connecting the strands and keeping them
aligned
 Removing the grey (Grecian
Formula)
• Active constituent is lead acetate
• Reacts with the disulfide links in keratin to
produce what black compound?
• Also does some structural damage
Animal hair protein composition
• Sheep’s wool: also the fibrous protein
keratin, but with high glycine & tyrosine
content
Do you want change a bad hair day?
To a Good Hair day?
Perm(?) – have your keratin 1o structure modified

HSCH2COOH

H2O2
 Use some Protein Chemistry on
your hair!
• Slightly basic solution of thioglycolic acid is used:
cleaves the disulfide links and makes new SH
bonds (reset hair)
• Then Dilute! Peroxide used in final Oxidation step
of “perm” (otherwise bleaching effect!)

• Covalent S-S bonds in new positions give

permanent structure (recall : position of the
disulfide linkages is part of 1o structure)
 Hydrogen bonding and your hairdo
• Hydrogen bonds N-H....O=C Between
adjacent strands of fibrous protein are much
weaker than the S-S covalent bonds, but
there are many more hydrogen bonds,
which form a large part of hair structure
• Hence excess water will break these up and
permit restructuring of hair upon drying
• Water not strong enough to break S-S bonds
 Hair gels

• First ingredient is water

• Contain “protein mimics”
• Water miscible copolymers with low
melting points
• Dimethylaminomethacrylate
Coloured gels
 Protein mimics in hair gels
• Y=N , thus an amide ; EO & PO are
polymer chains
 Protein Denaturation
• Heat
• Mechanical agitation
• pH change
• Inorganic salts
• polar organic solvents
• Soaps and detergents
 Heating of protein causes
denaturation
• Frying eggs
• Cooking meat-insoluble collagen protein is
converted into soluble gelatin to be used in
Jello, gravy, or glue (from horses)
 Mechanical Agitation
• Beating egg whites-proteins denature at the
surface of the air bubbles
• Cream of tartar (the dipotassium salt of
tartaric acid) is added to beaten egg whites
to keep them stiff for mousse and meringue
preparation, by raising the pH
 Disinfection by denaturation
• Ethanol acts via denaturation of bacterial
protein
• Detergents and soaps disrupt association of
protein sidechains of bacterial protein
 Protein Denaturation: Origin of
Cheese?
• Arab merchant carrying milk across the
desert in a pouch made from sheep’s
stomach
• Action of heat caused milk to form a watery
liquid and a soft curd with a “pleasing taste”
• Rennet containing the enzyme Rennin in the
sheep’s stomach caused curd formation
 Sour milk , Cheese
• Increased amount of lactic acid (from fermentation
of lactose by lactobacillus bacteria) causes lower
pH
• Induces protein denaturation and then coagulation
• Casein proteins make up 80% of protein in skim
milk
• Precipitation of casein by low pH results in curds,
essential to cheese making
 Macronutrients in Cheese
• Protein ~ 30% (variable); Brie 20%,
Cheddar 25%, Parmesan 40%
• Fat 25-35%
• Carbohydrates (sugars) 0.1-1%
• ---------------------------------------------
• Water content variable, but up to ~35%
• **Cottage cheese 79% water, 17% protein,
3% carb, 0.3% fat
 Cottage Cheese
• Easy to make (in your cottage!)
• Is just the unripened curd from skim milk
• Most of the fat is removed before the
clotting process, hence high protein to fat
rato: low (<1% fat content)
• If add cream can get fat content up to 2-6%,
(cream cottage cheese)
 Cream cheese
• Also unripened (like cottage cheese) but it
is made from a mixture of milk and cream
• High fat content(> 30%)
 Swiss (Emmental) Cheese
• A hard cheese ripened by bacteria
producing CO2, thus forming holes

• Processed cheese –a blend of several

(mostly cheddar). Components are mixed,
melted and reformed
 Yogurt
• From fermentation of milk (generally skim)
using 2 microorganisms only, Lactobacillus
bulgaris and Streptococcus thermophilus.
• Prior to innoculation with these bacteria,
milk is heating to boiling to kill all other
microorganisms
• Yogurt itself can be used for innoculation
 Probiotic Yogurt
• Promotes a the growth of a healthy balance
of ~200 types of bacteria in the GI tract
• A very healthy breakfast food!
• Promotes regularity
• Live cultures of lactobacillus and l.
bulgaricus are best
 Proteins by Structure
Proteins

Simple Conjugated

Fibrous Globular Lipo- Glyco- Hemo-

insoluble soluble
‘structural’ ‘reactive’
hair, horn enzymes HDL, interferon hemo-
LDL globin
 Proteins by Structure

Fibrous

Collagens Elastins Keratins Myosins

bones lungs hair/feathers muscles

tendons ligaments horn/nails
cartilage
 Proteins by Structure

Globular

Albumins Globulins

egg whites antibodies(-globulin)

enzymes
 Proteins by Function

Enzymes - the biological catalysts

Contractile - muscle
Hormones - insulin, growth hormone
Neurotransmitters - endorphins
Storage - store nutrients, eg. seeds,
casein in milk
Transport - hemoglobin
Structural - collagen, keratins
Protective - antibodies
Toxins - snake venom, botulinum
 Protein - Daily Requirements
Average adult contains ~10kg of protein;
~300g is replaced daily by recycling and intake.
We need to take in *
~70g of high quality protein or ~80g of lower quality

* this varies with age, size and energy demand,eg.

infants: 1.8g/kg/day
children: 1.0g/kg/day
adults: 0.8g/kg/day
Recommended: ~15% of daily Caloric intake
Normally the body does not store proteins. Since
they are the major source of nitrogen they are
constantly being broken down and reconstructed.

Protein is lost in urine, fecal material, sweat,

hair/nail cuttings and sloughed skin.
 (Non)Essential Amino Acids
The essential amino acids (10) are those that
our bodies cannot synthesize. We must obtain
them from our dietary intake.
They are:
histidine, isoleucine, leucine, lysine,
methionine, phenylalanine, threonine,
tryptophan, valine (and arginine in infants).

The non-essential a.a.(10) can be synthesized in our

bodies from breakdown products of metabolism.
 Tryptophan: a sleep inducer?
• Tryptophan - present in turkey
• Lots of anecdotal evidence re: sleep
inducing effects of a turkey dinner!
• Any connection between tryptophan and
serotonin? (present in the brain) ; deemed to
act as a calming agent and hence plays a
role in sleep induction
Serotonin from tryptophan
 Tryptophan as a “nutraceutical”
• Foods (macronutrients) acting to have a
pharmaceutical effect

• Nutr aceutical
 Tryptophan therapy
• For sleep disorders
 Other foods with tryptophan

Milk, cheese, soy products

Also, avoid caffeine for at least 5 hours before
bed
 Vegetarian Diets
• Main challenge is to get enough high
quality protein with the correct balance of
essential amino acids
Protein Content (approx.%) of Foods
cheese 30
peanuts 27
chicken 21
fish 18
beef 18
soy 17
wheat 13
beans 7
rice 8
peas 7
milk 6
corn 4
cassava 3
potatoes 2
Complete or High - Quality protein contains all
the essential amino acids in about the same
ratio as they occur in human protein.
eg. meat, fish, poultry

Incomplete or Low-quality protein is deficient

in one or more of the essential amino acids.
eg. protein from plant sources.
Essential Amino Acids – Meat vs. Veg
 Note: Tryptophan levels
• Dates contain high levels !
• Also milk
 Complementary proteins are combinations of
incomplete or low-quality proteins that taken
together provide about the same ratio of
essential amino acids as do high-quality
proteins.

In general: Legumes(peas/beans) + Grains

Most of the people of the world depend on grains,

not meat, as their major source of proteins. Many
of these people have developed food combinations
containing complementary proteins that allow them
to live without suffering from malnutrition.
Some, mainly meat-free, food combinations that
produce a diet with complementary protein.

‘Continent’ Staple Diet

Asia Rice + Soy
S. America Beans + Corn
Middle East Hummus(Chick peas) +
Bulgar wheat/Pita bread
India Lentils + Yoghurt +
Unleavened bread
N. America Peanut Butter Sandwich
 Malnourished - the inability to obtain sufficient
complete protein, ie. essential amino acids,
for the body to function properly.

Symptoms - extreme emaciation, bloated

abdomen, lack of pigmentation, mental
apathy, eventual death, eg. no antibodies,
muscle breakdown, capacity of brain
diminished ( increases from ~350g at
birth to full size(~1200g) by 2 yrs).

1 of every 8 people on Earth suffers malnutrition severe

enough to stunt physical and mental growth.
 Digestion
Digestion is the breakdown of ingested foods by
hydrolysis (catalyzed by enzymes) into relatively
small molecules, eg. simple sugars, amino acids,
fatty acids/glycerol, that can be absorbed through
the intestinal walls and into the circulatory system.

Starch - begins in mouth, stops in stomach, finishes

in small intestine
Triglycerides - primarily in small intestine
Protein - begins in stomach, completed in small
intestine
The Liver - the Nutrient Bank of the Body
After digestion most food nutrients pass to the
liver for distribution, storage and conversion.

Glucose - broken down/oxidized for energy, build

glycogen, directed to bloodstream to
nourish cells
Amino acids - form enzymes, sent to cells to build
protein, oxidized for energy.
Digestion:
In one end
and out the
other.
 Daily Caloric Intake
NA averages Source Recommended

>40% Total Fat 30%

>20% Sat. Fat 10%
35% *Total Carbo’ >55%
>15% Sugars ~12%
25% Protein <15%
Cholesterol 300mg
~4.0g Sodium 2.4g
<12g *Dietary Fibre 23g
 Food Composition > % by Weight
Food Water Protein Fat Carbo Cal/100g
let/tom/beans 92 1.5 0.2 4.5 24
pot/car/corn 80 2.5 0.3 20 80
soy 74 10 5 10 118
rice 70 2.5 0.5 26 119
white bread 36 9 3 50 269
fruits 84 1 0.5 14 53
berries 85 1 0.5 12 60
nuts 5 15 65 15 630
 Food Composition > % by Weight
Food Water Protein Fat Carbo Cal/100g
lean meat 65 25 8 0 175
chicken 71 24 4 0 136
salmon 64 27 7 0 182
whole milk 87 4 4 5 65
cot. cheese 79 17 0.3 3 86
cheddar 37 25 32 2 398
eggs 74 13 13 1 163
 Problem Set #4
• Chapt 17# 1,3,4,5,11,14,15,22
 CHEMISTRY of COOKING
• Why is my toast brown?
• What happens to meat when it is cooked?
• What causes the odour of roasted meats?
• What causes the flavour of roasted coffee?
• Does cooking introduce harmful
byproducts?
 WHY COOK AT ALL?
• It tastes (and smells) good
• Food is made more digestible and allows us
to eat a greater range of food
• Releases the raw materials that we might
otherwise not be able to digest (ie. in meat)
• Cooking destroys bacteria such as
salmonella, E coli etc., thus more safe
 Let’s compare
• Grilling (BBQ) gas or with hard wood
coals: char broiled
• Oven broiled
• Oven roasting (baked)
• Boiled
• Steamed
• Microwave
Your thoughts?????
 What is known for sure
• BBQ (Grilling) of high fat content meats at
high temp produces Polynuclear aromatic
hydrocarbons (PNAH’s)
• Planar molecules: intercalate into the major
groove of the DNA double helix
• Can be cancer inducing if DNA directed
synthesis of protein gets out of control
 First cancerous lesions
• Observed in chimney sweeps in UK in
1700’s and workers in coal tar industry
• Exposure to soot on skin
• PNAH’s common in soot from burning of
coal
Some structures of PNAH’s
 That BBQ steak flavour
• 15 different PNAH’s have been isolated from the
outer layer of charcoal broiled steak
• 8 micrograms of 1,2-benzopyrene per kilo of steak
• Arise from decomposition of fat that drips on to
the glowing charcoal and the subsequent
vaporization of the hydrocarbons and deposition
on the surface of the meat
 Other possible carcinogens
• Heterocyclic amines (HCA’s) added to list
of known carcinogens in 2005
• Arise from reaction of creatine (an amino
acid found in muscle) and carbohydrate
• Higher temps from grill, frying or oven
broiling increase the concentrations
 How to minimize these risks
• Use lean meats or remove fat
• Cook at lower temperatures (ie allow coals
to cool to embers if using hardwood) or on a
gas grill move food to an upper rack
• Use marinades (olive oil or citrus based)
• Avoid overcooking
 Marination: also denaturation
• Long time (days) exposure to acid (in
vinegar) will denature some protein and
tenderize some meat
• Also kills Salmonella, but not E coli
 Cooking fish on the grill
• Leave skin on and do most of the grilling
skin side down–easily separated when fish
is cooked
• Use cedar grilling planks to impart a rich
smoky flavour –keeps fish moist and no
charring
Happy BBQ’s (a summer tradition)

• Should be a treat, not every night ! Enjoy!

 In general
• Long slow cooking (baking, roasting) are
best due to lower temperatures used
• Minimizes formation of potential
carcinogens
• BUT, real dangers from undercooked food
containing harmful bacteria (E coli: can be
fatal almost immediately)
• Hamburger (large surface area) and poultry
Let’s think positively about cooking!

• Where do those wonderful aromas come

from (ie. baking bread), coffee brewing,
cookies from the oven and the traditional
Sunday roast beef dinner (with oven
browned potatoes , carrots etc)
Traditional (for some)
 The Maillard browning reaction
• Louis-Camille Maillard investigated this
~1910
• Reaction between an amino acid (in protein)
and a sugar (from starch)
• Accelerated in a basic environment: amino
group becomes non protonated
 What is the mechanism?
• The N atom of the amino acid is
nucleophilic (ie it is seeking a partially
positive target)
• The C=O in the open form of sugars
(aldohexoses and aldoketoses) ie glucose
and fructose has a partially positive C, due
to the fact that O is more electronegative
than C
 Nucleophilic addition
• Lone pair of electrons on N “attacks”
partially positive C of C=O group

• N: C=O
 What are the products ?
• Reaction occurs around 300F
• Biscuit, popcorn, bread, tortilla flavour
(odour threshold is 0.06 ng/L)
 Can you name this compound?
• Probably not!

• 2-acetyl-3,4,5,6-tetrahydropyridine

• Oops-it is a heterocyclic amine (HCA)

 Acrylamide structure
• Planar, thus a potential DNA intercalator
 Note the similarity to acrylamide
structure
• So……….could acrylamide be generated by
the Maillard reaction?

• Yes! (J. Agr. Food Chem. 53, 4628-4632

(2005)
 Glucose reacts with Asparagine
• N attacks C=O !
 Many steps later..a bit of …
• Exact mechanism unknown for acrylamide
formation
• Many other products!
 What about acrylamide?
• No evidence yet of human cancer induction
• Considered a probable carcinogen based on
animal studies
• First noted in Swedish study in 2002
• Particularly in deep fried foods
• Low levels suggest not a critical issue
Maillard reactions of Tryptophan
• In turkey: reacts with glucose to produce a
glycoside
Tryptophan: glycoside
Maillard reaction in roasted nuts
• Almost all nuts are roasted before
consumption; kills bacteria and increases
flavor
• Peanut roasting has been studied in detail
due to widespread allergies
• Allergy is protein induced
• Some of the Maillard products may increase
the allergenicity of peanuts
 Summary
• Maillard reaction produces hundreds of
compounds which are responsible for
pleasant odour and taste of protein
containing foods.
• It also produces trace amounts of
acrylamide (the price we pay for flavor!)
 Other Browning reactions
• Maillard “browning” often accompanied by
carmelization if more carbohydrate (sugar)
is present
• Carmelized onions: both protein and sugar
naturally present in the onion (Demo).
• Heating of sucrose alone can cause
carmelization (browning) (Demo)
 Cooking meat: the ultimate protein
denaturation!
• Browning of meat with flour before stewing
• Maillard reaction between protein of the
meat and the starch of the flour
 Structure of Meats
• Beef: red meat is mostly muscle: contains bundles
of fibrous proteins, held together by a natural
“glue” which is mostly collagen
• Meat is “lubricated” with pads of fat which act to
cushion the muscle
• Carving meat: go across the fibres-cuts them into
shorter lengths, easier to chew and digest
• Fish muscle has shorter fibres and is more delicate
and cooks at a much lower temperature
 Effect of Heat on meat structure
• Protein strands shrink and tangle and
squeeze out the fat, which has now melted
• Increased temperature causes proteins to
tangle more and meat gets tougher and
smaller
• Colour changes: myoglobin (red) turns gray
when the denatured hemochrome forms
 Poultry: red and white meat
• Red meat is in muscles –ie legs and wings
due to presence of myoglobin
• White meat in breasts: not used for exercise,
hence no oxygen carrying myoglobin is
needed
 Dinner anyone????
• Club sandwich
 Or……for vegetarians
• Vietamese delight
 How about some jello for
dessert?
• Essential ingredients : gelatin –a protein
extracted from collagen (present in
connective tissue of farm animals)
• Water
• Sugar (or artificial sweetener)
• Food colouring
 Wild berry jello (Demo)
• Blue colour comes from anthocyanins
(natural) in grape skins, cabbage
• Elasticity is due to triple helix structure of
the protein gelatin
• Typical sequence has AlaGlyPro ArgGly…
• Made via dissolution of gelatin powder in
hot water, then addition of equal volume of
cold, then refrigerate (~2 hours to form)
 Can I make it with added fresh
pineapple?
• No
• Pineapple contains the enzyme Bromelain
(also a protein) which degrades the gelatin
into its constituent amino acids, which then
dissolve
• Fresh Kiwi fruit causes the same effect
 Is sugar required?
• No: can be made with artificial sweeteners
 Food colourings
• Red cabbage extracts
• Anthocyanins-a class of compounds also
found in skins of grapes etc.
• Added to many foods –natural colouring
agent
• pH dependent
• Can be used as an indicator
Chemical structure of
anthocyanins
 Jams
• Do not contain gelatin, but rather pectin: a
soluble carbohydrate based fiber found in
apples,plums and citrus fruits
• Blueberries, strawberries, cherries-low
pectin content, need to add pectin
• Require sugar and acid (lemon juice) for
gel formation and for thickening
• No elasticity!!