10. Amino acidsProteins

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10. Amino acids/Proteins Chapter 17
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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.
  MW10,000

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Amino Acids - Protein building blocks
An amino acid is a compound having both a
carboxyl group(-COOH) and an amino group(-NH2).
All amino acids from protein

have the -NH2 attached at the C ?
to the COOH (as well as the H- R-).
H H2N C COOH R
All naturally occurring ?-amino acids, except
glycine (RH), are chiral and the L
stereoisomer.
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H H2N C COOH R
There are 20 ?-amino acids 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).
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Amino Acids - 1
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Amino Acids - 2
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Amino acids

• Contain both an acidic functional group (COOH)
  and a basic one (-NH2), NH or N
• Thus reactions are highly pH dependent

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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)

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Leucine ionic forms

• Cation below pH 2.4
• Neutral between pH 2.4 and 9.6
• Anionic above pH 9.6

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Leucine zwitterion

• pH2.4
• pH

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Peptides Buildup/Breakdown
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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

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Tripeptides

• Consider amino acids Glycine (G), Alanine (A) and
  Phenylalanine (P)
• How many different tripeptides are possible if
  each amino acid must be present?

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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

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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.
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Some Protein Sequences
Oxytocin contracts smooth muscle
(induces labour)
Phe - Gln Tyr Asn Cys Cys - S-S
Pro - Arg - Gly
Ile - Gln Tyr Asn Cys Cys - S-S
Vasopressin - diuretic
Pro - Leu - Gly
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Val- Glu Gln
Ile-Gly
Insulin (21 30)
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-C
ys-Gly
Glu Arg Gly Phe
Leu His Gln-
Asn-Val-Phe
Thr-Lys-Pro-Thr-Tyr-Phe-
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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

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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

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Secondary (20) Structure - sheets
H bond
sheets/strands, eg. fingernails, silk
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Secondary Structure(20) - the ?-Helix
H-bonding - intramolecular
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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

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Dispersion forces

• Attractive when nuclei are separated by the sum
  of their van der Waals radii

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Tertiary Structure (30) - braids globs
collagen
hemoglobin
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Quaternary structure of proteins

• When a protein has 1 polypeptide chain, then
  interchain (between chains) interactions can
  occur
• Hemoglobin has this

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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.

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Myoglobin Structure
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To summarize

• Myoglobin cannot have quaternary structure since
  it has only one polypeptide chain
• Hemoglobin has 4 polypeptide chains and possesses
  quaternary structure

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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

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Active Site of Enzymes
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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)
the prion proteins
causing mad cow disease in animals or
Creutzfeldt-Jakob disease in humans may be caused
by a change/denaturation from a helical(water
soluble) form to a sheet (water insoluble)
form.
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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

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Protein in Human hair

• Keratin (fibrous protein) has the S containing
  amino acid cystine (1418) .
• S-S bonds (disulphide linkages) between cystine
  units give hair its strength by connecting the
  strands and keeping them aligned

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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

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Animal hair protein composition

• Sheeps wool also the fibrous protein keratin,
  but with high glycine tyrosine content

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Do you want change a bad hair day?
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To a Good Hair day?
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Perm(?) have your keratin 1o structure modified
HSCH2COOH
H2O2
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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)

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Hydrogen bonding and your hairdo

• Hydrogen bonds N-H....OC 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

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Hair gels

• First ingredient is water
• Contain protein mimics
• Water miscible copolymers with low melting points
• Dimethylaminomethacrylate

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Coloured gels
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Protein mimics in hair gels

• YN , thus an amide EO PO are polymer chains

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Protein Denaturation

• Heat
• Mechanical agitation
• pH change
• Inorganic salts
• polar organic solvents
• Soaps and detergents

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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)

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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

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Disinfection by denaturation

• Ethanol acts via denaturation of bacterial
  protein
• Detergents and soaps disrupt association of
  protein sidechains of bacterial protein

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Protein Denaturation Origin of Cheese?

• Arab merchant carrying milk across the desert in
  a pouch made from sheeps 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
  sheeps stomach caused curd formation

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Sour milk , Cheese , Yogurt

• 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

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Cheese Production

• Hard cheeses (Cheddar, Stilton,
  Gruyere)-microbial action reduces milk pH to 5.5.
  Bacteria ripens (ages) these.
• Addition of rennin (enzyme from calve stomach)
  causes hydrolysis of casein to give the curds
  (solid) and the whey (liquid)
• NB. Cheese manufactured using fungal rennin is
  marketed as vegetarian

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Roquefort and Blue Cheese

• Mold P.roquefortii is added to unripe cheese
• Roquefort is made from sheep milk
• Blue is made from cows milk

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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

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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 but very low (content)
• If add cream can get fat content up to 2-6,
  (cream cottage cheese)

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Cream cheese

• Also unripened (like cottage cheese) but it is
  made from a mixture of milk and cream
• High fat content( 30)

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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

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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

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Proteins by Structure
Proteins Simple
Conjugated 
Fibrous Globular Lipo- Glyco-
Hemo-
insoluble soluble
structural
reactive  hair, horn enzymes HDL,
interferon hemo-

LDL globin
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Proteins by Structure
Fibrous   Collagens Elastins
Keratins Myosins
bones lungs
hair/feathers muscles tendons
ligaments horn/nails
cartilage
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Proteins by Structure
Globular    Albumins
Globulins
egg whites
antibodies(?-globulin)

enzymes
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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
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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
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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.
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(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.
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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

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Serotonin from tryptophan
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Tryptophan as a nutraceutical

• Foods (macronutrients) acting to have a
  pharmaceutical effect
• Nutr aceutical

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Tryptophan therapy

• For sleep disorders

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Other foods with tryptophan

• Milk, cheese, soy products
• Also, avoid caffeine for at least 5 hours before
  bed

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Vegetarian Diets

• Main challenge is to get enough high quality
  protein with the correct balance of essential
  amino acids

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Protein Content (approx.) of Foods
cheese peanuts chicken fish beef soy wheat
beans rice peas milk corn cassava potatoes

30 27 21 18 18 17 13 7 8 7 6 4 3 2
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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.
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Essential Amino Acids Meat vs. Veg
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Note Tryptophan levels

• Dates contain high levels !
• Also milk

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Protein Equiv. Foods vs. Human Milk(35g)
Food Limit. aa. Milk Digest
Food(kg)
wheat corn rice beans soy potato cassava
eggs meat cow milk
lys try/lys lys val met/cys leu
met/cys leu try met/cys
81 72 52 51 44 72 82 37 43 44
79 60 75 78 78 74 60 97 97 97
13 8 8 24 34 2 1 13 22 3
.77 1.5 .92 .27 .17 4.6 12.5 .30 .21 1.4
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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.
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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
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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.
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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
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The Liver - the Nutrient Bank of the Body
After digestion most food nutrients pass to the
liver for distribution, storage and conversion.
broken down/oxidized for energy, build
glycogen, directed to bloodstream to
nourish cells
Glucose -
form enzymes, sent to cells to build
protein, oxidized for energy.
Amino acids -
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Digestion In one end and out the other.
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Daily Caloric Intake
NA averages Source
Recommended
Total Fat Sat. Fat Total Carbo
Sugars Protein Cholesterol Sodium
Dietary Fibre
40 20 35 15
25 4.0g
30 10 55 12 2.4g 23g
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Food Composition by Weight
Food Water Protein Fat Carbo
Cal/100g
let/tom/beanspot/car/corn soy
rice white bread fruits
berries nuts
92 80 74 70 36 84 85 5
1.5 2.5 10 2.5 9 1 1
15
0.2 0.3 5 0.5 3 0.5 0.5 65
4.5 20 10 26 50 14 12 15
24 80 118 119 269 53 60 630
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Food Composition by Weight
Food Water Protein Fat Carbo
Cal/100g
lean meat chicken salmon whole milk cot.
cheese cheddar eggs
65 71 64 87 79 37 74
25 24 27 4 17 25 13
8 4 7 4 0.3 32 13
0 0 0 5 3 2 1
175 136 182 65 86 398 163
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End of material for quiz 1

• Feb 9, 2008 at 1 PM in Southam Hall
• Calculators allowed

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Problem Set 4

• Chapt 17 1,3,4,5,11,14,15,22

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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?

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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

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In the news this week

• Jamie Morris (2 bonus) Ottawa Citizen Article
• Fries, cookies etc. may increase womens cancer
  risk because carbohydrate rich foods that are
  baked, grilled , roasted or toasted at over 120oC
  contain acrylamide, a known cancer inducing
  substance
• Ovarian and endometrial cancer

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The bigger question is..

• Is there any difference in how we cook our foods
  with respect to the formation of cancer causing
  compounds in general???
• What do you think?

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Lets compare

• Grilling (BBQ) gas or with hard wood coals char
  broiled
• Oven broiled
• Oven roasting (baked)
• Boiled
• Steamed
• Microwave

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Your thoughts?????
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What is known for sure

• BBQ (Grilling) of high fat content meats at high
  temp produces Polynuclear aromatic hydrocarbons
  (PNAHs)
• 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

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First cancerous lesions

• Observed in chimney sweeps in UK in 1700s and
  workers in coal tar industry
• Exposure to soot on skin
• PNAHs common in soot from burning of coal

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Some structures of PNAHs
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That BBQ steak flavour

• 15 different PNAHs 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

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Other possible carcinogens

• Heterocyclic amines (HCAs) 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

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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

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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

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Cooking fish on the grill

• Leave skin on and do most of the grilling skin
  side downeasily separated when fish is cooked
• Use cedar grilling planks to impart a rich smoky
  flavour keeps fish moist and no charring

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Happy BBQs (a summer tradition)

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

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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

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Lets 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)

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Traditional (for some)
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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

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What is the mechanism?

• The N atom of the amino acid is nucleophilic (ie
  it is seeking a partially positive target)
• The CO 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

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Nucleophilic addition

• Lone pair of electrons on N attacks partially
  positive C of CO group
• N CO

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What are the products ?

• Reaction occurs around 300F
• Biscuit, popcorn, bread, tortilla flavour (odour
  threshold is 0.06 ng/L)

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Can you name this compound?

• Probably not!
• 2-acetyl-3,4,5,6-tetrahydropyridine
• Oops-it is a heterocyclic amine (HCA)

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Acrylamide structure

• Planar, thus a potential DNA intercalator

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Note the similarity to acrylamide structure

• So.could acrylamide be generated by the
  Maillard reaction?
• Yes! (J. Agr. Food Chem. 53, 4628-4632 (2005)

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Glucose reacts with Asparagine

• N attacks CO !

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Many steps later..a bit of

• Exact mechanism unknown for acrylamide formation
• Many other products!

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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

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Maillard reactions of Tryptophan

• In turkey reacts with glucose to produce a
  glycoside

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Tryptophan glycoside
116
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

117
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!)

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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)

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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

120
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

121
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

122
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

123
Lunch anyone????

• Club sandwich

124
Orfor vegetarians

• Vietamese delight