10. amino acids/proteins chapter 17

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).

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 (R=H), are chiral and the ‘L’ stereoisomer.

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).

H

H2N C COOH

R

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

H2N CH

CH3

C

O

OH H N CH

H

C

O

OH

H2N CH

CH3

C

O

N CH

H

C

O

OH

H

H

+

alanine (ala) glycine (gly)

+ H2O

Peptide or amide linkage

amine end

alanylglycine (ala-gly) -a dipeptide

carboxylic acid end

dehydration

hydrolysis

Peptides – Buildup/Breakdown

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

Tyr Asn

Cys Cys -

S-S

Pro - Arg - Gly

Ile - Gln

Tyr Asn

Cys Cys -

S-S

Pro - Leu - Gly

Oxytocin – contracts smooth muscle (induces ‘labour’)

Vasopressin - diuretic

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

Cys-Thr-Ser-Ile

Val-

Glu

Gln

Cys Cys-Ser-Leu-Tyr-Gln-Leu-Glu-Asn-Tyr-Cys-Asn

Leu

His

Gln-

Glu

Arg

Gly

PheThr-Lys-Pro-Thr-Tyr-Phe-

Ile-Gly

Asn-Val-Phe

Insulin (21 + 30)

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

sheets/strands, eg. fingernails, silk

NN

O R

O

H

NN

OR

O

H

H

HH – bond

H-bonding -intramolecular

Secondary Structure(20) - the -Helix

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 

insoluble soluble ‘structural’ ‘reactive’ 

hair, horn enzymes HDL, interferon hemo-

Fibrous Globular Lipo- Glyco- 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,

Transport - hemoglobin Structural - collagen, keratins Protective - antibodies Toxins - snake venom, botulinum

casein in milk

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.

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.

They are:

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 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

Incomplete or Low-quality protein is deficient in one or more of the essential amino acids.

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

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.

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.

In general: Legumes(peas/beans) + Grains

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.

broken down/oxidized for energy, build glycogen, directed to bloodstream to nourish cells

form enzymes, sent to cells to build protein, oxidized for energy.

Glucose -

Amino acids -

Digestion:

In one end and out the other.

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

<12g

30% 10% >55% ~12% <15%

300mg 2.4g

23g

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

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

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!!