Proteins/Amino Acids

1. Proteins/Amino Acids

2. Preliminary Concepts • Protein is the principal constituent of organs and soft structures of the animal body • a continuous supply is needed from food sources throughout life for growth/repair • food protein  body protein • food protein: plant or animal • unique proteins found in each animal • no two are alike in physiological behavior

3. Roles of Protein • Bulk composition of the body (structural aspects of the cell) • oxidative metabolism (used as energy source in energy-deficient diets) • enzymes (globular proteins that regulate and influence metabolism) • plasma proteins (circulating, mobile proteins such as immune bodies) • hormones (regulatory role)

4. Proteins within Cell Wall

5. Special Functions • peptides (formation of proteins) • purines/pyrimidines (control of protein synthesis) • histamines (active compounds, allergies) • conjugated proteins (assist in the excretion of other compounds) • pigments (e.g., melanin, derived from amino acids)

6. Elementary Composition of Proteins • Chemical composition: primarily carbon, hydrogen, oxygen • additional difference: contain a fairly constant amount of nitrogen found in amino groups (17%) • many also contain sulfur, phosphorus and iron • structure is typically complex, having high molecular weight

7. Protein Classification • simple proteins: essentially pure proteins, when hydrolyzed, produce individual amino acids (e.g., egg albumin) • conjugated: protein unit linked to another non-protein unit (e.g., casein, the protein component of milk with phosphorus esterified to it via the AA serine (ser) • derived: modified proteins such as peptides, modified by heat, acidification,etc.

8. Conjugated Proteins • nucleoproteins: protein + nucleic acid (e.g., seed germs) • glycoproteins: protein + COH group (e.g., mucus) • phosphoproteins: protein+ P-containing compound (e.g., casein) • hemoglobins: protein + hematin or similar substance • lecithoproteins: protein + lecithin (e.g., fibrinogen)

9. Structure of Protein Molecule • As mentioned, proteins are sequences of amino acids hooked together by the amino group of one to the carboxyl group of another • this bond is known as the peptide linkage • AA found in protein are known as residues • protein chains of AA have typically 100-200 residues • many proteins have more than one chain

10. The Peptide Linkage

11. Protein Structure • primary: the sequence of AA’s forming the protein • secondary: forces generated by the close proximity of one AA residue to another (e.g.,  helix design or  pleated sheet)(i.e., certain amino acids can form bonds with others, if close enough, cysteine) • tertiary: bending of one AA chain due to attraction of individual AA’s distant from each other • quaternary: packing of chains together

12. Protein Structure

13. Amino Acids (AA) • As mentioned, proteins are polymerized residues of amino acids • the number and proportion of AA vary from protein to protein • when proteins are denatured, the AA remain • to study protein, you must study AA • at least 30 different AA, some essential others non-essential

14. Characterizing AA • Most AA are derived from lower or short-chain fatty acids (FA; such as acetic, proprionic or butyric acid) • naturally-occurring have L-configuration • synthetic have large proportion of D configs • soluble in water, amphoteric • show various types: aliphatic, aromatic, heterocyclic, etc.

15. “D” vs. “L” Configuration

16. Aliphatic Amino Acids

17. Aromatic Amino Acids

18. Chemical Determination of Protein • The direct determination of protein in tissue is impractical due to quantity/variation • nitrogen, however, occurs at fairly constant levels: • [N] x 6.25 = protein level • some proteins have well-known nitrogen levels (e.g., milk @ 15.7% N) • determined by Kjehldal N methodology

19. Protein/AA Quality • Amino acids are basically divided into two nutritional categories: • essential: those the animal cannot synthesize in sufficient quantity to support maximum growth, typically dietary in nature • nonessential: synthesized by animal body, typically non-dietary in nature • determined first by Rose (1930) working on factorial deletion with rats

20. Essential AA Exceptions • lysine (LYS) • arginine (ARG) • methionine (MET) • histidine (HIS) • isoleucine (ILE) • leucine (LEU) • threonine (THR) • tryptophan (TRY) • phenylalanine (PHE) • 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

21. Protein/AA Quantitative Requirements • A protein requirement is really an EAA requirement (why?) • reports persist on “protein” requirement • protein “requirement” for fish: 25-50% • the above statement says nothing about requirement: it doesn’t measure intake • unfortunately, not all sources of protein are “balanced”, not all are digestible • why? Variance due to culture conditions

22. Factors Affecting Protein “Requirement” • Size of fish/shrimp • water temperature • feed allowance/feeding rate • amount of non-protein energy sources • quality of protein (AA) • availability of extrinsic sources of nutrition • salinity (affects digestibility) • physiological/nutritional state

23. Additional Protein Requirement Info • 3 g catfish require up to 4x more protein intake on daily basis vs. 250 g catfish • pond sources of protein are typically protein dense (over 50% protein on DM basis) • protein “requirement” can be reduced by feeding more frequently w/attractant • NPU for most aquatics is around 40% • could vary with enzyme activity, molt status in crustaceans

24. Requirements for Amino Acids • Somewhat variable due to “apparent” nature of determinations • no standardized methodology can be applied due to differences in feeding behavior, treatment system design, way in which EAA is presented, etc. • for fish, the EAA requirements are similar to those of other animals • only difference is with ARG (Table 2.4, Lovell)

25. EAA Requirements of Several Fishes, Chickens and Swine AminoChannelTilapia AcidCatfishniloticaChicken Swine ARG4.34.25.6 1.2 HIS1.51.71.4 1.2 ILE2.63.13.3 3.4 LEU3.53.45.6 3.7 LYS5.15.14.7 4.4 MET+ CYS2.33.23.3 2.3 PHE + TYR5.05.75.6 4.4 THR2.03.63.1 2.8 TRY0.51.00.9 0.8 VAL3.02.83.4 3.2

26. Requirements for EAA • Requirement for one EAA can be partially mitigated by a NEAA • example: CYS sparing of MET • CYS replaces about 60% of MET • often reported as MET-CYS requirement • example: TYR sparing of PHE (about 50%)

27. Requirement for Lysine by Fish • Sciaenops ocellatus 4.43% • Oreochromis aurea 4.30% • Oncorhyncus tshawytscha 5.00% • Ictalurus punctatus 5.00% • Dicentrarchus labrax 4.82% • Morone saxatilis 3.4-4.0% • Cyprinus carpio 5.70%

28. EAA Requirements by Shrimp • the quantitative requirement for only two essential amino acids has been determined for shrimp: ARG, LYS • difficulty: crustaceans are sloppy eaters, don’t effectively use crystalline sources, experimental conditions allow cannibalism, extrinsic sources of EAA (bacteria) • difficult to formulate reasonable diet and vary only one EAA

29. Crystalline Amino Acids • Most EAA requirement studies have used CAAs • CAAs produced by bacteria • can help reduce formulation cost of feeds because they are 99% digestible • for most aquatics, no more than 12.5% of AA-N • problems: reduced palatability, leaching, rapid uptake

30. Amino Acid Metabolism: protein synthesis • Complex process occuring in most animal tissues • involves DNA, RNA and ribosomes • chromosomal DNA is storeplace of genetic information, transmission from one generation to the next • DNA = 4 nucleotides: adenine, guanine, cytosine, thymine

31. Protein Synthesis • DNA controls formation of RNA • one form of RNA (RNAt) transfers amino acids to ribosomes • ribosomes are the source of protein synthesis (anabolism) • protein synthesis (about 50 seconds/protein) • amino acids also catabolized for energy: transamination or oxidative deamination

32. Protein Digestibility • Dietary protein quality is determined by its bioavailability to the animal • “bioavailability” is not simply digestibility, it also includes assimilation and incorporation of the AA into protein • most common index of protein bioavaila- bility is apparent protein digestibility (ADP) • ADP = % of protein not rejected as feces

33. Protein Digestibility • APD depends upon degree of purity of proteins involved • purity ratings: purified, semi-purified, practical • purified: gelatin, casein, soy-isolate • semi-purified: hi-pro soybean meal, glutens • practical: fish meal, squid meal, peanut meal, rice bran, etc.

34. Protein Digestibility • Contrary to popular beliefs, animal protein is not more digestible than plant protein • digestibility really determined by level of purification and degree of interaction (competition for absorption sites) between one nutrient and another • factors: salinity (indirect), size/age (indirect)

35. Amino Acid Digestibility • apparent amino acid digestibility (AAAD) is directly related to protein digestibility • proteins vary in APD, but amino acids don’t in terms of AAAD (proteins compete with other nutrients, AA’s don’t) • amino acids are typically absorbed in the gut (fish) and midgut/midgut gland (shrimp) • 6 transport mechanisms: 1) neutral AA’s (mono’s), 2) basic (diamino’s), 3) acidic (dicarboxylic’s), 4) aromatics, 5) alanine and 6) glycine

36. Amino Acid Assimilation • “assimilation” is not transport, it involves the appearance of AA’s in various tissues (blood, hemolymph, muscle, etc.) • appearance OK for intact-sourced AA’s, but rapid and unsynchronized for CAA’s (too much, too quickly) • CAA’s possibly used with increased feeding frequency

37. Amino Acid Toxicity/Antagonism • Toxicity/antagonisms are result of dietary imbalances in EAA • when one EAA is fed in excess it can also increase the requirement for another, structurally-similar EAA • toxicity = overfeeding of one EAA and negative effects not mitigated by increasing other EAA • antagonism = one EAA regulates uptake of another • LEU/ILE in catfish (Robinson, 1984) • LYS/ARG in shrimp (Fox, 1992)