Proteins/Amino AcidsProteins/Amino Acids

Preliminary ConceptsPreliminary Concepts

Protein is important in building of Protein is important in building of organs and soft structures of the animal organs and soft structures of the animal bodybody

A continuous supply is needed from A continuous supply is needed from feed sources throughout life for feed sources throughout life for growth/repairgrowth/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

Roles of ProteinRoles of Protein bulk compositionbulk composition (structural parts of the cell) (structural parts of the cell)

oxidative metabolismoxidative metabolism (used as energy source) (used as energy source)

enzymesenzymes (regulate and influence metabolism) (regulate and influence metabolism)

plasma proteinsplasma proteins (circulating immune bodies) (circulating immune bodies)

hormoneshormones

Proteins within Cell Proteins within Cell WallWall

Elementary Elementary Composition of ProteinsComposition of ProteinsChemical compositionChemical composition: primarily carbon, : primarily carbon,

hydrogen, oxygenhydrogen, oxygen

additional differenceadditional difference: nitrogen in amino : nitrogen in amino groups (~17%)groups (~17%)

may contain sulfur, phosphorus and ironmay contain sulfur, phosphorus and iron

structure is complexstructure is complex

Structure of Protein Structure of Protein MoleculeMolecule

Proteins are sequences of amino acids hooked Proteins are sequences of amino acids hooked together by the amino group (NHtogether by the amino group (NH33)of one to the )of one to the carboxyl group (COOH) of another called a carboxyl group (COOH) of another called a peptide bond.peptide bond.

Protein chains of AA have typically 100-200 Protein chains of AA have typically 100-200 residuesresidues

Many 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 proximity : forces generated by the close proximity

of one AA residue to another (e.g., of one AA residue to another (e.g., helix design or helix design or pleated sheet)(i.e., certain amino acids can form pleated sheet)(i.e., certain amino acids can form bonds with others, if close enough, cysteine)bonds with others, if close enough, 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 when proteins are denatureddenatured, the AA , 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 made using short-chain Most AA are made using short-chain fatty acids (FA; such as acetic, 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

““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

AA QualityAA Quality

Amino acids divided into two groups:Amino acids divided into two groups:

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

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)

ser/gly essential for ser/gly essential for chickschicks

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

no big problem for no big problem for ruminants, why?ruminants, why?

All essentials are in “L” All essentials are in “L” form only humans really form only humans really need HISneed HIS

ExceptionsExceptions

Do we need protein?Do we need protein? A protein requirement is really an EAA A protein requirement is really an EAA

requirement (why?)requirement (why?) To say protein “requirement” for fish: 25-50% To say protein “requirement” for fish: 25-50%

says nothing about requirement: it doesn’t says nothing about requirement: it doesn’t measure intake. measure intake.

Why?Why? Variance due to culture conditions Variance due to culture conditions Unfortunately, not all sources of protein are Unfortunately, not all sources of protein are

“balanced”, not all are digestible.“balanced”, not all are digestible.

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/attractant (why?)feeding more frequently w/attractant (why?)

Net Protein Utilization (NPU) for most Net Protein Utilization (NPU) for most aquatics is around 40%aquatics is around 40%

could vary with enzyme activity, molt status could vary with enzyme activity, molt status in crustaceansin crustaceans

Requirements for Amino Requirements for Amino Acids?Acids?

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

no standardized methodologyno standardized methodology can be applied 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 animals (all similar???)those of other animals (all similar???)

major difference is with major 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

AminoAmino ChannelChannel TilapiaTilapia

AcidAcid CatfishCatfish niloticanilotica ChickenChicken SwineSwine

ARGARG 4.34.3 4.24.2 5.65.6 1.21.2

HISHIS 1.51.5 1.71.7 1.41.4 1.21.2

ILEILE 2.62.6 3.13.1 3.33.3 3.43.4

LEULEU 3.53.5 3.43.4 5.65.6 3.73.7

LYSLYS 5.15.1 5.15.1 4.74.7 4.44.4

MET+ CYSMET+ CYS 2.32.3 3.23.2 3.33.3 2.32.3

PHE + TYRPHE + TYR 5.05.0 5.75.7 5.65.6 4.44.4

THRTHR 2.02.0 3.63.6 3.13.1 2.82.8

TRYTRY 0.50.5 1.01.0 0.90.9 0.80.8

VALVAL 3.03.0 2.82.8 3.43.4 3.23.2

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%

Sparing EAAsSparing EAAs

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%)

EAA Requirements for EAA Requirements for Shrimp?Shrimp?

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

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

*Purified amino acids from bacterial culture.*Purified amino acids from bacterial culture.

Crystalline Amino Acids Crystalline Amino Acids (CAAs)(CAAs)

Most EAA requirement studies have used CAAsMost EAA requirement studies have used CAAs

CAAs produced by bacteria means pure!CAAs produced by bacteria means pure!

can help reduce formulation cost of feeds can help reduce formulation cost of feeds because they are 99% digestible (few; 75-85% in because they are 99% digestible (few; 75-85% in others)others)

problemsproblems: reduced palatability, leaching, rapid : reduced palatability, leaching, rapid uptakeuptake

Amino Acid Amino Acid Metabolism: Metabolism: protein protein

synthesissynthesis

Complex process occuring in most animal Complex process occuring in most animal tissuestissues

involving DNA, RNA and ribosomesinvolving DNA, RNA and ribosomes

chromosomal DNA is storeplace of genetic chromosomal DNA is storeplace of genetic information, transmission from one generation information, transmission from one generation to the nextto the next

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

Protein SynthesisProtein Synthesis DNA controls formation of RNADNA controls formation of RNA

tRNA transfers amino acids to ribosomestRNA transfers amino acids to ribosomes

ribosomesribosomes are the source of protein synthesis are the source of protein synthesis ((anabolismanabolism))

protein synthesis (about 50 seconds/protein) (fast!!)protein synthesis (about 50 seconds/protein) (fast!!)

amino acids also catabolized for energy amino acids also catabolized for energy

transaminationtransamination or oxidative or oxidative deaminationdeamination

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

most common index of protein bioavailability is most common index of protein bioavailability is apparent protein digestibilityapparent protein digestibility (APD) (APD)

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

Protein Protein DigestibilityDigestibility

APDAPD depends upon degree of purity of proteins depends upon degree of purity of proteins involvedinvolved

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 meal, : fish meal, squid meal, peanut meal, rice bran, etc.rice bran, etc.

Protein Protein DigestibilityDigestibility

Contrary to popular beliefs, animal Contrary to popular beliefs, animal protein is protein is notnot more “digestible” than more “digestible” than plant proteinplant protein

digestibility really determined by level of digestibility really determined by level of purificationpurification and degree of interaction 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 directlydirectly related to protein digestibilityrelated to protein digestibility

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

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

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

Amino Acid Amino Acid AssimilationAssimilation

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

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

CAA’s possibly used with increased feeding CAA’s possibly used with increased feeding frequencyfrequency

Amino Acid Amino Acid Toxicity/AntagonismToxicity/Antagonism

Toxicity/antagonisms are result of dietary Toxicity/antagonisms are result of dietary imbalancesimbalances in EAA in EAA

when one EAA is fed in when one EAA is fed in excessexcess it can also it can also increaseincrease the requirement for another, the requirement for another, structurally-similar EAAstructurally-similar EAA

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

antagonismantagonism = one EAA regulates uptake of = one EAA regulates uptake of another 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)