Chapter 9

Protein and Amino Acids

• Protein vary widely in chemical composition, physical property, size shape, solubility, biological function.

General structure of amino acid

• R is the remainder of the molecule attached to the C atom associated with the α-amino group of the amino acid.

NH2 O

R —— C ——C

OH OH

• Amino acids not synthesized in animal tissues of most specie in sufficient amounts to meet are termed essential or indispensable, whereas those generally not needed in the diet because of adequate tissue synthesis are termed nonessential or dispensable.

Essential• Arginine• Histidine• Isoleucine• Leucine• Lysine• Methionine• Phenylalnine• Threonine• Tryptophen• Valine

Nonessential• Alanine• Aspartic acid• Citrulline• Cystine• Glutamic acid• Glycine• Hydroxyproline• Proline• Serine• tyrosine

• Arginine: is required for some species for maximum growth, adult do not require. However dog, cat show severe sign of deficiency.

• Glutamine: is a dispensable, 但生病時 , 是唯持正常代謝所需，是 indispensable.

• In most animal:

• in mucosa : glutamate ＋ proline 合成 ornithine ， ornithine 轉成 citrulline ，再送至 腎臟產生 arginine( urea cycle)

• In cat 、 dog: ornithine→citrulline 與 citrulline→arginine 未能進行，∴ arginine is essential 。在 higher protein ，缺 arginine 無法進行 urea cycle ，引起 hyperammonia

• Taurine : 1. Synthesized by most mammals from methionine an

d cysteine2. It is indispensable for cat only.3. a amino sulfonic acid, occurs as a free amino acid. i

s not present in protein4. develop degeneration of retina of the eye.(myocardi

um and retina contain high concentration of free taurine)

5. Taurine deficiency occurred in the low dietary sulfer-containing amino acids

• Methionine→ cysteine→

cysteine sulphinic acid →hypotaurine

→taurine

Structure of protein

1. Primary structure = linkage between α-carboxyl of one amino acid group of another (peptide linkage)

R

C

H

H 2N

O

C N

H

C C OH

H

OR1

2. Secondary structure = polypeptide in the form of a α-helix by means of hydrogen bond CO—HN. α—helix or β—pleated sheet, supercoil

3.Tertiary structure :

polypeptide into a globular form by means of:1. hydrophobic

2. disulfide linkage (s-s)

3. salt bridge

4. hydrogen bonding

a globular form

4. Quaternary structure

alignment of several tertiary structure into one protein by means of hydrogen bonds, electrostatic, salt bonds

Ex: hemoglobin consist of four single strand tertiary.

• Gastrointestinal microflora synthesize protein from nonprotein N sources.

• The length of the chain and the order of arrangement of amino acids within the chain are two of the main factors determining the characteristics of the protein.

• Synthesis of protein from amino acids. The linkage between amino acids called peptide bond. Elongation of chain from tripeptide, polypeptide.

• All naturally occuring amino acid are in the L-form. which is the biologically active form. ( 但合成大多為 L 與 D form 的混合物 ).

• All proteins can be classified on the basis of their shape; their solubilities in water, salt, acids, bases and alcohol; and other special characteristics.

• Egg albumin is the most perfect protein for meeting animal need because of its nearly ideal amino acid composition and its high digestibility.

• Corn endosperm: Zein( 玉米蛋白 ) is low in lysine and tryptophan. Opaque-2 corn is lower contain in zein, 此種玉米含較高量之 lysine 及 tryptophan.

• A sensitive measure of the nutritive value of protein is the balance among its essential balance, no two protin have an identical amino acid composition.

A.

Globular protein 球狀蛋白Albumin

Globulin

Glutelin

Prolamines

Histone

Protamine

B.

Fibrous protein

Collagens

Elastins

Keratin

C.

Conjugated protein

Lipoprotein

• Represented by the menbrane proteins of animal cells.

• myelin: in nervous system mucolipid erythrocyte membrane

• The protein content of plasma lipoprotein ranges from 2% in chylomicron to about 50% in high density lipoprotein.

Glycoprotein

• Chondroitin sulfate A. B. C. (protein complex of sulfated polysaccharides) 出現在 cartilage, tendon and skin.

• Mucoprotein: complex of protein with: – amino sugar (glucosamine, galactosamine.)– hexose, manose, galactose.– pentose, fucose– sialic acid

Glycoprotein

• serum cholinesterase

• gonadotrophin( 性腺素 )

• mucous secretion

• ovalbumin (mucoprotein containing glucosamine, mannose).

• ovomucoid (containing hexosamine, hexose; trypsin inhibitor in egg white)

• aspartic acid ( 是主要與醣類結合之胺基酸 )

Functions

• Present as components of cell membrane, muscle, skin, hair, hooves, blood plasma protein, enzyme, hormones, immune antibody.

Tissue protein1. Collagen: triple helix, 含 proline, hydroxylproli

ne

2. Elastin: resemble denatured collagen and consist of long, randomly ordered, ploy-peptide chain. minor component of musculature.

3. myofibrilar protein : the proteins of sacroplasm.( 肌肉與稀鹽類均質後可萃取﹐含有 20 種酵素 )

4. contractile protein : three protein- actin, tropomyosin B and myosin. 與 muscle contraction有關 .

Tissue protein

5. Keratins : proteins of hair, wool, feathers, hooves, horns, claws, beaks.

6. Blood proteins : albumin and a series of globulins, apoprotein

7. Enzymes : hydrolytic and degradative metabolic and synthetic reaction.

8. Hormones : insulin, growth hormone, gonadotrophic hormone, parathyroid hormone and calcitonin 。

Metabolically active peptides and polypeptides

• A large number of peptides and poly-peptide been identified in modulating growth and other metabolic activity.

• such as – insulin-like growth factor (IGF-1,)– transforming growth factor beta (TGF-B)– fibroblast growth factor (FGF)– nerve growth factor (NGF)

Metabolism• two phases: catabolism (degradation) and

anabolism (synthesis)• The conversion of dietary protein to tissue protein

involves:1. Intact dietary protein

hydrolysis in GI tract (catabolism)

2. Amino acid in intestinal lumen

absorption from GI tract

3. Amino acid in blood

synthesis in tissues (anabolism)

4. Intact tissue proteins

• Even protein hydrolyzed easily in the GI tract do not have a high nutritional value, if they have a deficiency or an unbalance of one or more amino acids.

• Dietary protein not containing the proportion of essential amino acids to meet the animal needs cannot be used efficiently for tissue protein synthesis.

Digestion

• Protein→→→ tripeptide, dipeptide, amino acid( lumen) → absorption → brush border enzyme (dipeptidase, tripeptidase) →

dipeptidase, tripeptidase in enterocycte

→ amino acids

Hydrolysis determine the degree of absorption of amino acid

(A) Digestible protein refer to that disappearing from the ingesta as it passes down the GI tract.

– Nitrogen in feces include:1. Unabsorbed dietary N .2. Metabolic fecal N

Metabolic fecal N : 1.Normal metabolism of tissue protein cells 2.sloughed residue of digestive enzyme. 3.other substrate secreted into the lumen.

(B) After absorption, amino acid are subject to further losses in utilization through metabolism in the liver and other tissues. losses N mainly as urinary urea N (mammal), or uric acid (bird).

(C) The degree of utilization of feed protein depend on1. digestibility, absorbability

2. utilization of its component amino acids after absorption.

Absorption of amino acids1. early postnatal life:

– absorbed by pinocytosis. ( 如 immune globulins and lactoferrin)

2. postnatal life:– absorbed by active transport ( two system )

• one for neutral amino acid.• one for basic amino acid.

– 相關性質之 amino acid 由於競爭 carrier, 吸收會受抑制。如 leucine 與 isoleucine methionine 與 lysine.

3. peptide are absorbed directly from the rumen and omasum

Fate of amino acids after absorption

1. tissue protein synthesis

2. synthesis enzyme, hormone and metabolites

3. deamination or transamination and use of the carbon skeleton for energy.

• Amino acid in the GI have three source:

• 1.Dietary ingestion

• 2.recycle from other nitrogen substance( digestive enzyme, sloughed mucosa cell)

• 3.synthesized by microorganism

A. Synthesis by microorganism

• Bacteria, protozoans can synthesis all amino acids in presence of ammonia, S, carbon sources

• Rumen, large intestine• In ruminant, synthesis amino acid from non

protein nitrogen.B. Body tissueTissue synthesis and degradation of amino a

cid in animal. Muscle, liver, brain, adipose tissue and others…..

Sites of amino acid degradation

• Microbes, cell and tissue specific pathways degrade all amino acids,

• Liver is the principle organ, and small intestinal cells to process of degradation.

• Transamination, decarboxylation, hydroxylation .

• Product of degradation: amionia, S, fatty acids (VFA). CO2

Nitrogen cycling in the intestine

• Dietary nitrogen→intestine →blood

• Endogenous N (urea) →intestinal lumen →microbial action →NH3 →used for incorporation into amino acids to produce microbial protein →digested by host

NO in animal nutrition and health

• Nitric oxide is a mediator of immune function, a neurotransmitter, a signaling molecule, and endothelium-derived relaxing factor and other positive role in metabolism.

• It is a cytotoxic free radical.• Is synthesized from arginine through action of th

e enzyme nitric oxide synthase (NOS).• Many nutrient, including protein and amino acid

may modulate NO production by NOS.

Ruminant nitrogen metabolism

• RUP: rumen undegraded protein

Synthesis of protein

DNA (chromosomal component of cell)

│ carry the genetic information

↓

transcript into a strand of messenger RNA

↓

tranlation of mRNA into protein

Whole body protein turnover

• Is a dynamic process involving continuous and simultaneous protein synthesis and protein degradation.

• Each organ or tissue has its own rate of protein turnover.

• The rate of body muscle growth is affected by the rate of protein synthesis relative to degradation.

Deamination and Transamination

• Deamination :removal of the amino group from carbon of amino acid and entrance of the amino group into the urea.

• Transamination: transfer an amino group from one amino acid to the carbon of a keto acid.

Urea cycle

• The urea or ornithine cycle is a key metabolic phenomenon in protein metabolism.

• Uric acid is end product of purine metabolism in human and other primate, in other mammals the principal end product is the oxidation product of uric acid, allantoin.

Protein and amino acid requirement and deficiency

1. Nonruminant (pig, chichken, human) require essential amino acid.

2. Nonruminant herbivores, and adult ruminant depend on nonprotein N and synthesize protein or amino acid.

3. Adult ruminant (cattle, sheep) can depend entirely on nonprotein N in the diet by virtue of their rumen microbes.

• Because most energy sources are low in protein and protein supplement are expensive, so inadequate protein is the most common.

• protein is diverted to energy only when it is provide in excess of the metabolic requirement or calorie intake is insufficient.

sign of protein deficiency

• anorexia,• reduced growth rate• negative N balance• reduced feed efficienc

y• reduced serum protei

n concentration• anemia

• fat accumulation in the liver

• edema• reduced birth weight• reduced milk

production• reduced enzyme,

hormone synthesis

• Individual amino acid deficiency result in deamination of the remaining amino acids, loss of NH3 as urea, and use of the carbon chain for energy.

• Ex: tryptophan deficiency : eye cataracts.

threonine, methionine : fatty liver

lysine (in bird) : abnormal feathering

• individual feedstuffs are inadequate amino acid

• Ex: corn: lysine, tryptophane,

SBM: rich in lysine, tryptophan, deficient in methionine-cystine.

seasame meal: inadequate lysine

• first limit amino acid: lysine

• second and third limit amino acid: threonine, tryptophan

D-amino acid and nonprotein N

• Natural: L-form.

• D-form amino acid used inefficiency. (except of methionine).

• Nonprotein nitrogen (NPN)

=diamonium citrate, Urea, amino acid, peptide, amines, amide and nucleic acid ( 存於forage).

• The efficiency of utilization of NPN compounds depend on

1. solubility of the NPN.

2. availability to the microflora of readily available carbohydrate.

Antagonism

• growth depression can be overcome by supplementation with an amino acids structurally similar to the antagonist

• Ex: lysine and Arginine. (structure similar)

• Excess of lysine→ growth depress →improve by addition of arginine

• Antagonist differ from imbalance of amino acids

Toxicity

• adverse effect of an amino acid in excess cannot be overcome by supplementation with another amino acid

• Ex: Methionine

Imbalance

• proportion of dietary amino acids that has an adverse effect presentable by a relatively small amount of the most limiting amino acid.

• animals will reject the imbalanced diet, but will support life.

protein-free-diet will be consumed largely, but not support life.

Excessive protein intake

• Depression in weight gain, feed intake. Hair dull and coarse.

• Ammonia toxicity (urea excessive)

Measure of nutritive value of proteins

1. Biological value (BV) =

• BV is defined as that percentage of N absorbed from the GI tract is available for productive body function.

• estimate of the efficiency of use of the absorbed protein for combined maintenance and growth.

N intake - ( fecal N + urinary N )x100%

N intake - fecal N

2. protein efficiency ratio (PER) =

body weight gain (g)

protein consumed (g)

3. Net protein Utilization (NPU) =

(body N with test protein

)

– (body N with

protein-free diet)

x100%total N intake

4. Net protein value (NPV) =

BV × digestion coefficient.

5. Assay of free amino acid concentration of blood plasma :

• changing in amino acid patterns following ingestion of the test protein.

6. Blood urea N:

• At level of dietary protein either above or below the requirement or with diet deficient or imbalanced in one or more amino acid, blood urea N is elevated. (serum urea fit well)

• All of the estimates of protein utilization described have their limitations and no one estimate is superior to all others under all conditions.

• PER is the best estimate of protein value for growth.

Biological availability of amino acids

1. microbiological assay :

• test material is digested by enzyme, acid or alkaline hydrolysis, and the amino acid composition of hydrolysate is determined by microbiological assay

• the rate and degree of release of amino acid is taken as an index of the availability to the animal.

2. fecal analysis• Amino acid availability (%) =

AA total － ( TFAA protein － TFAA protein-free )

AA total

3. analysis of terminal ileum content :

• absorption of amino acid from the large intestine and degree of degradation in the lower intestine ileum are not defined well.

• amino acid content of ileum contents can be used to calculate the availability of individual amino acid.

4. growth assay :

• comparing the growth curve of animals fed the test protein with that of animals fed the amino acid diet containing the amino acids of concern.

• Estimate the proportion of amino acid in the test protein utilized for growth.

5. plasma free amino acids :

• The change in relative concentration of amino acids in the plasma following a meal of the test protein.

• The duration of fasting and the selection of appropriate intervals for blood sampling are important.

• Processing methods including grinding, pellecting, drying, oil extraction, and heating.

• The greatest single factor affecting amino acid availability from feedstuffs is proper heating of feedstuffs during processing.

• application of heat must be a balance between beneficial and destructive effects.

• destruct or delayed release during digestion owing to change in linkage between amino acids and other components.