Proteins/Amino Proteins/Amino AcidsAcids

Preliminary ConceptsPreliminary Concepts

Protein is the principal constituent of Protein is the principal constituent of organs and soft structures of the animal organs and soft structures of the animal bodybody

a continuous supply is needed from food a continuous supply is needed from food sources throughout life for growth/repairsources throughout life for growth/repair

food protein food protein body protein body protein food protein: plant or animalfood protein: plant or animal unique proteins found in each animalunique proteins found in each animal no two are alike in physiological behaviorno two are alike in physiological behavior

Roles of ProteinRoles of Protein Bulk compositionBulk composition of the body (structural of the body (structural

aspects of the cell)aspects of the cell) oxidative metabolismoxidative metabolism (used as energy (used as energy

source in energy-deficient diets)source in energy-deficient diets) enzymesenzymes (globular proteins that regulate (globular proteins that regulate

and influence metabolism)and influence metabolism) plasma proteinsplasma proteins (circulating, mobile (circulating, mobile

proteins such as immune bodies)proteins such as immune bodies) hormoneshormones (regulatory role) (regulatory role)

Proteins within Cell Proteins within Cell WallWall

Special FunctionsSpecial Functions

peptidespeptides (formation of proteins) (formation of proteins) purines/pyrimidinespurines/pyrimidines (control of protein (control of protein

synthesis)synthesis) histamineshistamines (active compounds, allergies) (active compounds, allergies) conjugated proteinsconjugated proteins (assist in the (assist in the

excretion of other compounds)excretion of other compounds) pigmentspigments (e.g., melanin, derived from (e.g., melanin, derived from

amino acids)amino acids)

Elementary Elementary Composition of ProteinsComposition of Proteins

Chemical compositionChemical composition: primarily carbon, : primarily carbon, hydrogen, oxygenhydrogen, oxygen

additional differenceadditional difference: contain a fairly : contain a fairly constant amount of nitrogen found in amino constant amount of nitrogen found in amino groups (17%)groups (17%)

many also contain sulfur, phosphorus and many also contain sulfur, phosphorus and ironiron

structure is typically complex, having high structure is typically complex, having high molecular weightmolecular weight

Protein Protein ClassificationClassification

simple proteinssimple proteins: essentially pure proteins, : essentially pure proteins, when when hydrolyzedhydrolyzed, produce individual amino , produce individual amino acids (e.g., egg albumin)acids (e.g., egg albumin)

conjugatedconjugated: protein unit linked to another : protein unit linked to another non-protein unit (e.g., non-protein unit (e.g., caseincasein, the protein , the protein component of milk with phosphorus component of milk with phosphorus esterified to it via the AA serine (ser)esterified to it via the AA serine (ser)

derivedderived: modified proteins such as : modified proteins such as peptides, modified by heat, acidification,etc.peptides, modified by heat, acidification,etc.

Conjugated Conjugated ProteinsProteins

nucleoproteinsnucleoproteins: protein + nucleic acid : protein + nucleic acid (e.g., seed germs)(e.g., seed germs)

glycoproteinsglycoproteins: protein + COH group (e.g., : protein + COH group (e.g., mucus)mucus)

phosphoproteinsphosphoproteins: protein+ P-containing : protein+ P-containing compound (e.g., casein)compound (e.g., casein)

hemoglobinshemoglobins: protein + hematin or similar : protein + hematin or similar substance substance

lecithoproteinslecithoproteins: protein + lecithin (e.g., : protein + lecithin (e.g., fibrinogen)fibrinogen)

Structure of Protein Structure of Protein MoleculeMolecule

As mentioned, proteins are sequences of As mentioned, proteins are sequences of amino acids hooked together by the amino amino acids hooked together by the amino group of one to the carboxyl group of another group of one to the carboxyl group of another

this bond is known as the this bond is known as the peptide linkagepeptide linkageAA found in protein are known as AA found in protein are known as residuesresiduesprotein chains of AA have typically 100-200 protein chains of AA have typically 100-200

residuesresiduesmany proteins have more than one chainmany proteins have more than one chain

The Peptide The Peptide LinkageLinkage

Protein StructureProtein Structure primaryprimary: the sequence of AA’s forming the protein: the sequence of AA’s forming the protein secondarysecondary: forces generated by the close : forces generated by the close

proximity of one AA residue to another (e.g., proximity of one AA residue to another (e.g., helix helix design or design or pleated sheet)(i.e., certain amino acids pleated sheet)(i.e., certain amino acids can form bonds with others, if close enough, can form bonds with others, if close enough, cysteine)cysteine)

tertiarytertiary: bending of one AA chain due to attraction : bending of one AA chain due to attraction of individual AA’s distant from each otherof individual AA’s distant from each other

quaternaryquaternary: packing of chains together : packing of chains together

Protein StructureProtein Structure

Amino Acids (AA)Amino Acids (AA)

As mentioned, proteins are polymerized As mentioned, proteins are polymerized residues of amino acidsresidues of amino acids

the number and proportion of AA vary the number and proportion of AA vary from protein to proteinfrom protein to protein

when proteins are denatured, the AA when proteins are denatured, the AA remainremain

to study protein, you must study AAto study protein, you must study AA at least 30 different AA, some essential at least 30 different AA, some essential

others non-essentialothers non-essential

Characterizing AACharacterizing AA

Most AA are derived from lower or short-Most AA are derived from lower or short-chain fatty acids (FA; such as acetic, chain fatty acids (FA; such as acetic, proprionic or butyric acid)proprionic or butyric acid)

naturally-occurring have naturally-occurring have L-configurationL-configuration synthetic have large proportion of synthetic have large proportion of D D

configsconfigs soluble in water, amphotericsoluble in water, amphoteric show various types: aliphatic, aromatic, show various types: aliphatic, aromatic,

heterocyclic, etc.heterocyclic, etc.

““D” vs. “L” D” vs. “L” ConfigurationConfiguration

Aliphatic Amino Aliphatic Amino AcidsAcids

Aromatic Amino Aromatic Amino AcidsAcids

Chemical Chemical Determination of Determination of

ProteinProtein

The direct determination of protein in tissue The direct determination of protein in tissue is impractical due to quantity/variationis impractical due to quantity/variation

nitrogen, however, occurs at fairly constant nitrogen, however, occurs at fairly constant levels:levels:

[N] x 6.25 = protein level[N] x 6.25 = protein level some proteins have well-known nitrogen some proteins have well-known nitrogen

levels (e.g., milk @ 15.7% N)levels (e.g., milk @ 15.7% N) determined by Kjehldal N methodologydetermined by Kjehldal N methodology

Protein/AA QualityProtein/AA Quality

Amino acids are basically divided into two Amino acids are basically divided into two nutritional categories:nutritional categories:

essentialessential: those the animal cannot : those the animal cannot synthesize in sufficient quantity to support synthesize in sufficient quantity to support maximum growth, typically dietary in naturemaximum growth, typically dietary in nature

nonessentialnonessential: synthesized by animal body, : synthesized by animal body, typically non-dietary in naturetypically non-dietary in nature

determined first by Rose (1930) working on determined first by Rose (1930) working on factorial deletion with ratsfactorial deletion with rats

Essential AAEssential AA

lysine (LYS)lysine (LYS) arginine (ARG)arginine (ARG) methionine (MET)methionine (MET) histidine (HIS)histidine (HIS) isoleucine (ILE)isoleucine (ILE) leucine (LEU)leucine (LEU) threonine (THR)threonine (THR) tryptophan (TRY)tryptophan (TRY) phenylalanine (PHE)phenylalanine (PHE) valine (VAL)valine (VAL)

dogs, rats

ser/gly essential for chicks

pigs = rats (max growth)

pigs don’t need ARG, HIS, LEU for maintenance

no big problem for ruminants, why?

All essentials are in L form

only humans really needs HIS

Exceptions

Protein/AA Quantitative Protein/AA Quantitative RequirementsRequirements

A protein requirement is really an EAA A protein requirement is really an EAA requirement (why?)requirement (why?)

reports persist on “protein” requirementreports persist on “protein” requirementprotein “requirement” for fish: 25-50%protein “requirement” for fish: 25-50% the above statement says nothing about the above statement says nothing about

requirement: it doesn’t measure intake requirement: it doesn’t measure intake unfortunately, not all sources of protein are unfortunately, not all sources of protein are

“balanced”, not all are digestible“balanced”, not all are digestiblewhy?why? Variance due to culture conditions Variance due to culture conditions

Factors Affecting Factors Affecting Protein “Requirement”Protein “Requirement”

Size of fish/shrimpSize of fish/shrimp water temperaturewater temperature feed allowance/feeding ratefeed allowance/feeding rate amount of non-protein energy sourcesamount of non-protein energy sources quality of protein (AA)quality of protein (AA) availability of extrinsic sources of nutritionavailability of extrinsic sources of nutrition salinity (affects digestibility)salinity (affects digestibility) physiological/nutritional statephysiological/nutritional state

Additional Protein Additional Protein Requirement InfoRequirement Info

3 g catfish require up to 4x more protein 3 g catfish require up to 4x more protein intake on daily basis vs. 250 g catfishintake on daily basis vs. 250 g catfish

pond sources of protein are typically protein pond sources of protein are typically protein dense (over 50% protein on DM basis)dense (over 50% protein on DM basis)

protein “requirement” can be reduced by protein “requirement” can be reduced by feeding more frequently w/attractantfeeding more frequently w/attractant

NPU for most aquatics is around 40%NPU for most aquatics is around 40% could vary with enzyme activity, molt status could vary with enzyme activity, molt status

in crustaceansin crustaceans

Requirements for Requirements for Amino AcidsAmino Acids

Somewhat variable due to “apparent” nature Somewhat variable due to “apparent” nature of determinationsof determinations

no standardized methodology can be applied no standardized methodology can be applied due to differences in feeding behavior, due to differences in feeding behavior, treatment system design, way in which EAA is treatment system design, way in which EAA is presented, etc.presented, etc.

for fish, the EAA requirements are similar to for fish, the EAA requirements are similar to those of other animalsthose of other animals

only difference is with only difference is with ARGARG (Table 2.4, Lovell) (Table 2.4, Lovell)

EAA Requirements of EAA Requirements of Several Fishes, Chickens Several Fishes, Chickens

and Swineand Swine

Amino Channel Tilapia

Acid Catfish nilotica Chicken Swine

ARG 4.3 4.2 5.6 1.2

HIS 1.5 1.7 1.4 1.2

ILE 2.6 3.1 3.3 3.4

LEU 3.5 3.4 5.6 3.7

LYS 5.1 5.1 4.7 4.4

MET+ CYS 2.3 3.2 3.3 2.3

PHE + TYR 5.0 5.7 5.6 4.4

THR 2.0 3.6 3.1 2.8

TRY 0.5 1.0 0.9 0.8

VAL 3.0 2.8 3.4 3.2

Requirements for EAARequirements for EAA

Requirement for one EAA can be Requirement for one EAA can be partially mitigated by a NEAApartially mitigated by a NEAA

exampleexample: CYS sparing of MET: CYS sparing of MET CYS replaces about 60% of METCYS replaces about 60% of MET often reported as MET-CYS often reported as MET-CYS

requirementrequirement exampleexample: TYR sparing of PHE (about : TYR sparing of PHE (about

50%)50%)

Requirement for Lysine Requirement for Lysine by Fishby Fish

Sciaenops ocellatus Sciaenops ocellatus 4.43% 4.43% Oreochromis aureaOreochromis aurea 4.30%4.30% Oncorhyncus tshawytschaOncorhyncus tshawytscha 5.00%5.00% Ictalurus punctatusIctalurus punctatus 5.00%5.00% Dicentrarchus labraxDicentrarchus labrax 4.82%4.82% Morone saxatilisMorone saxatilis 3.4-3.4-

4.0%4.0% Cyprinus carpioCyprinus carpio 5.70%5.70%

EAA Requirements by EAA Requirements by ShrimpShrimp

the quantitative requirement for only two the quantitative requirement for only two essential amino acids has been determined essential amino acids has been determined for shrimp: for shrimp: ARGARG, , LYSLYS

difficultydifficulty: crustaceans are sloppy eaters, : crustaceans are sloppy eaters, don’t effectively use crystalline sources, don’t effectively use crystalline sources, experimental conditions allow cannibalism, experimental conditions allow cannibalism, extrinsic sources of EAA (bacteria)extrinsic sources of EAA (bacteria)

difficult to formulate reasonable diet and difficult to formulate reasonable diet and vary only one EAAvary only one EAA

Crystalline Amino Crystalline Amino AcidsAcids

Most EAA requirement studies have used Most EAA requirement studies have used CAAsCAAs

CAAs produced by bacteriaCAAs produced by bacteriacan help reduce formulation cost of feeds can help reduce formulation cost of feeds

because they are 99% digestiblebecause they are 99% digestible for most aquatics, no more than 12.5% of for most aquatics, no more than 12.5% of

AA-NAA-Nproblemsproblems: reduced palatability, leaching, : reduced palatability, leaching,

rapid uptakerapid uptake

Amino Acid Amino Acid Metabolism: Metabolism: protein protein

synthesissynthesis

Complex process occuring in most Complex process occuring in most animal tissuesanimal tissues

involves DNA, RNA and ribosomesinvolves DNA, RNA and ribosomeschromosomal DNA is storeplace of chromosomal DNA is storeplace of

genetic information, transmission genetic information, transmission from one generation to the nextfrom one generation to the next

DNA = 4 nucleotides: adenine, DNA = 4 nucleotides: adenine, guanine, cytosine, thymineguanine, cytosine, thymine

Protein SynthesisProtein Synthesis DNA controls formation of RNADNA controls formation of RNA one form of RNA (RNAt) transfers amino one form of RNA (RNAt) transfers amino

acids to ribosomesacids to ribosomes ribosomes are the source of protein ribosomes are the source of protein

synthesis (synthesis (anabolismanabolism)) protein synthesis (about 50 protein synthesis (about 50

seconds/protein)seconds/protein) amino acids also catabolized for energy: amino acids also catabolized for energy:

transamination or oxidative deaminationtransamination or oxidative deamination

Protein Protein DigestibilityDigestibility

Dietary protein quality is determined by its Dietary protein quality is determined by its bioavailabilitybioavailability to the animal to the animal

““bioavailability” is not simply digestibility, it bioavailability” is not simply digestibility, it also includes assimilation and incorporation also includes assimilation and incorporation of the AA into proteinof the AA into protein

most common index of protein bioavaila- most common index of protein bioavaila- bility is bility is apparent protein digestibilityapparent protein digestibility (ADP)(ADP)

ADPADP = % of protein not rejected as feces = % of protein not rejected as feces

Protein Protein DigestibilityDigestibility

APDAPD depends upon degree of purity of depends upon degree of purity of proteins involvedproteins involved

purity ratingspurity ratings: purified, semi-purified, : purified, semi-purified, practicalpractical

purifiedpurified: gelatin, casein, soy-isolate: gelatin, casein, soy-isolate semi-purifiedsemi-purified: hi-pro soybean meal, glutens: hi-pro soybean meal, glutens practicalpractical: fish meal, squid meal, peanut : fish meal, squid meal, peanut

meal, rice bran, etc.meal, rice bran, etc.

Protein Protein DigestibilityDigestibility

Contrary to popular beliefs, animal Contrary to popular beliefs, animal protein is not more digestible than plant protein is not more digestible than plant proteinprotein

digestibility really determined by level of digestibility really determined by level of purification and degree of interaction purification and degree of interaction (competition for absorption sites) (competition for absorption sites) between one nutrient and anotherbetween one nutrient and another

factorsfactors: salinity (indirect), size/age : salinity (indirect), size/age (indirect)(indirect)

Amino Acid Amino Acid DigestibilityDigestibility

apparent amino acid digestibility (apparent amino acid digestibility (AAADAAAD) is ) is directly related to protein digestibilitydirectly related to protein digestibility

proteins vary in APD, but amino acids don’t in proteins vary in APD, but amino acids don’t in terms of AAAD (proteins compete with other terms of AAAD (proteins compete with other nutrients, AA’s don’t)nutrients, AA’s don’t)

amino acids are typically absorbed in the gut amino acids are typically absorbed in the gut (fish) and midgut/midgut gland (shrimp)(fish) and midgut/midgut gland (shrimp)

6 transport mechanisms6 transport mechanisms: 1) neutral AA’s : 1) neutral AA’s (mono’s), 2) basic (diamino’s), 3) acidic (mono’s), 2) basic (diamino’s), 3) acidic (dicarboxylic’s), 4) aromatics, 5) alanine and 6) (dicarboxylic’s), 4) aromatics, 5) alanine and 6) glycineglycine

Amino Acid Amino Acid AssimilationAssimilation

““assimilationassimilation” is not transport, it involves ” is not transport, it involves the appearance of AA’s in various tissues the appearance of AA’s in various tissues (blood, hemolymph, muscle, etc.)(blood, hemolymph, muscle, etc.)

appearance OK for intact-sourced AA’s, appearance OK for intact-sourced AA’s, but rapid and unsynchronized for CAA’s but rapid and unsynchronized for CAA’s (too much, too quickly)(too much, too quickly)

CAA’s possibly used with increased CAA’s possibly used with increased feeding frequencyfeeding frequency

Amino Acid Amino Acid Toxicity/AntagonismToxicity/Antagonism

Toxicity/antagonisms are result of dietary imbalances Toxicity/antagonisms are result of dietary imbalances in EAAin EAA

when one EAA is fed in excess it can also increase the when one EAA is fed in excess it can also increase the requirement for another, structurally-similar EAArequirement for another, structurally-similar EAA

toxicitytoxicity = overfeeding of one EAA and negative = overfeeding of one EAA and negative effects not mitigated by increasing other EAAeffects not mitigated by increasing other EAA

antagonismantagonism = one EAA regulates uptake of another = one EAA regulates uptake of another LEU/ILE in catfish (Robinson, 1984)LEU/ILE in catfish (Robinson, 1984) LYS/ARG in shrimp (Fox, 1992)LYS/ARG in shrimp (Fox, 1992)