Amino Acids and Proteins PDF

AMINO ACIDS AND PROTEINS Introduction to Proteins and Amino Acids  Building blocks of protein  Biologic activity of the protein  Growth, repair, and maintenance  About half of the 20 amino acids must be supplied  Classification of Amino Acids  At le...

AMINO ACIDS AND PROTEINS Introduction to Proteins and Amino Acids  Building blocks of protein  Biologic activity of the protein  Growth, repair, and maintenance  About half of the 20 amino acids must be supplied  Classification of Amino Acids  At least one of both amino and carboxylic acid functional group  Amino acids that have an amino group bonded directly to the alpha-carbon are referred to as alpha amino acids  Every alpha amino acid has a carbon atom, called an alpha carbon; bonded to a carboxylic acid – COOH group; and amino, -NH2 group; a hydrogen atom; and an R group that is unique for every amino acid Peptide Bonds  A peptide bond is the amide bond formed between carboxyl group of one amino aid and the amino group of another  Amino Acids  Essential amino acids: from diet  Nonessential amino acids: body can produce them via liver Essential Non Essential Valine Alaine Leucine Arginine (semi) Isoleucine Aspargine Methionine Aspartate Tryptophan Cystine Phenylalanine Glutamic Acid Threonine Glycine Lysine Ornithine Histidine Proline Serine Amino Acids  All are synthesized by proteolytic enzymes: Pepsin and Trypsin  These enzymes digest dietary proteins into their constituent amino acids The amino group is removed from amino acid by  Body breaks down amino acids to make proteins  Deamination: breaking down amino acids  Transamination: transfer of amino acids Aminoacidopathies  Aminoacidopathies are a class of inherited errors of metabolism ; deficiency of enzyme cannot prod other amino acids  Leads to excess of an amino acid; cannot convert or break down amino acid  Eg phenylalanine in excess can cause mental retardation  Phenylketonuria (PKU): lack of Phenylalanine hydroxylase  Occurs in approx. 1 of 15,000 births  Phenylalanine hydroxylase (PAH) breakdown phenylalanine  tyrosine  Phenylpyruvic acid builds up  brain damage  Screenings  Guthrie Bacterial Inhibition Assay (Bacillus subtilis): semiquantitative test; agar used has a growth inhibitor  Agar is inoculated with B. subtulis but it will not grow, blood sample containing phenylalanine will allow growth  Musty urine odor Aminoacidopathies  Tyrosinemia: type I is the most severe  tyrosine catabolism  Excretion of tyrosine and tyrosine catabolites in urine  Lack of Furmarylacetate hydrolase (type I)  Treatment: low protein diet and drug nitisinone  Kidney and liver failure  cirrhosis Alkaptonuria: Lack of homogentisate oxidase  About 1 in 250,000 births  arthritic symptoms later in life  Excess of homogentistate acid  Urine left at room temp turns black  Can develop arthritis  Treated with Vitamin C Aminoacidopathies  Maple Syrup Urine Disease (MSUD)  Autosomal recessive gene  Absence or reduced activity of alpha-ketoacid decarboxylase  Maple syrup, burnt sugar odor of the urine, skin and breathe  Appear normal at birth but then CNS symptoms : seizures, hypoglycemia, retardation  Babies can make leucine, isoleucine , valine  Modified Guthrie test at 450 nm and 360 nm wavelength Aminoacidopathies  Homocystinuria  Lacks enzyme cystathionine beta-synthase (metabolism of amino acid methionine)  Cannot break down methionine  Guthrie test neonatal testing with HPLC as confirmatory method  Untreated can lead to multisystemic disorder of connective tissue; muscles, CNS, bone weakness, thrombosis  Treatment: dietary restriction of methionine; vitamin B6 Aminoacidopathies  Cystinuria  Defect in amino acid transport system: renal tubules cannot reabsorb cysteine  Detected by cysteine in urine  Inadequate reabsorption Treatment: prevent formation of cystine stones by high fluid intake; can develop renal calculi  Penicillamine prescribed when high fluid intake does not stop stone formation\  Cianide nitroprusside test Structures of Proteins Primary sturtcure  Simplest lvl of protein structure: string of amino acids linked together  Starts from N terminal + carboxyl terminal C end  Eg: hemoglobin Structures of Proteins  Primary Structure  The simplest level of protein structure  The primary structure of a protein starts from the amino-terminal (N) end and ends in the carboxyl-terminal (C) end  Example: hemoglobin Structure of Proteins  Secondary Structure is repeating structures stabilized by hydrogen bonds between the amino acids within the protein.  Common secondary structures are the alpha- helix, beta-pleated sheet and turns, with most serum proteins forming a helix.  New properties such as strength and flexibility are added Structure of Proteins  Tertiary Structure refers to the overall shape, or conformation, of the protein molecule; 3 three- dimensional  At this stage they have functional properties  Can interact with side chains, form ionic bonds,and disulfide bonds  Physical and chemical properties depends on 3* stricture  Structure of Proteins  Quaternary Structure is the shape that results from the interaction of more than one protein molecule or protein subunits  Eg: hemoglobin Structures of Protein  When any of its structures are disturbed, protein loses its functionality and chemical characteristics  All structures can be degraded down to primary structure  Denaturation: leads to loss of function  Heat  Hydrolysis  Strong acid (pH)  Strong alkali (pH)  Enzymatic action  Urea exposure or other substance  Ultraviolet exposure  Heavy metals and solvents  Proteins are described by their content, charge, solubility, immunogenicity, and synthesis capability  All proteins contain carbon, hydrogen, oxygen, sulfur, and nitrogen  Carbons/lipids don’t have nitrogen   Proteins are described by:  Nitrogen content  Approximately 16% of serum protein is of nitrogen content  Charge  The reactive acid or basic group involved in the peptide linkage Proteins exist in 3 charge forms based on pH of the surrounding environment  pH at which an amino acid or protein has no charge (isoelectric point = pI)  Basic pH > pI : negatively charged  Acidic pH < pI : positively charged   Solubility  When charge on protein surface, protein is hydrophilic (loves water)  Immunogenicity  Effective antigen-antibody synthesis Proteins  Synthesis Capability  Helps balance the body chemistry (homeostasis)  Rate of synthesis varies and is based on the content  Enzymes Hormone receptors Functions Transporters of Proteins Immunoglobulins: proteins that protect against “foreign” invaders Structural  Storage Functions Energy source of Proteins continued.. Osmotic Force Tissue nutrition (unique to proteins)   Simple Proteins  Contain peptide chains composed of only amino acids  Ex: albumin, hemoglobin, immunoglobulins IgG, IgA, and IgM  Connective tissues, tendons, bone, and muscles Two Major  Fibrous: hydrophobic; structure (wool fibers repel water) Classification  Globular: hydrophilic; transporters, enzymes, cell communication  Conjugated Proteins s of Proteins  Consists of a protein and a nonprotein (prosthetic) group: none protein eg glycoprotein; carb + protein, lipoprotein Lipid + protein, copper + protein etc  The nonprotein group is the nonamino part of a conjugated protein  Ex: lipids, carbohydrates, porphyrins, metals, and others Plasma Proteins  Two groups:  Albumin 60% of total proteins  Globulins  There are four major types of globulins, each with specific properties and actions  Pre albumin, albumin; alpha 1,2 beta, gamma (glubulins)  All made in liver except gamma globulins (  A typical blood panel will provide four different measurements  1. Total protein= albumin+globulin  2. Albumin = total proteins – globulins  3. Globulins (total protein – albumin)  4. Albumin/Globulin (A/G) ratio: 1.1-2.5 (normal range)  Anything less than 1 is clinically significant for autoimmune disease, liver disease and kidney disease   Prealbumin (Transthyretin)  Transport protein for thyroxine and other thyroid hormones eg vitamin A  2 day half life (best for malnutrition diagnosis)  Migrates before albumin on electrophoresis  Decreased levels in Plasma  hepatic damage, Proteins  acute-phase inflammatory response  tissue necrosis  Increased levels in  patients receiving steroids  alcoholism  chronic renal failure Plasma Proteins  Albumin  Takes lead role in colloid osmotic pressure (COP) of the intravascular fluid  Decreased levels mean less water uptake  Increased levels more water maintained  Buffer pH  60% of total protein  negative acute-phase reactant protein: decreased concentration during inflammation  Transports thyroid hormone and water  Half life of 17 days  Albumin  Decreased levels are associated with:  Hereditary analbuminemia (autosomal recessive trait where albumin is not produced)  Bisalbuminemia  two albumin bands instead of one on electrophoresis  Glomerulonephritis  Malnutrition; low protein diet Plasma   Liver disease Proteins Muscle waste  Inflammation  Increased  Severe dehydration  Excessive IV albumin therapy Plasma Proteins  Albumin Methods  Dye-binding methods (most widely used method; absorbance measured)  Dyes that selectively bind only to albumin  Methyl orange  Nonspecific for albumin; Beta lipoproteins, and some alpha 1 and alpha 2 globulins will bind  Bromcresol Green (BCG)  Interferences: hemoglobin (for every 100 mg/dL, albumin increased by 0.1 g/dL)  Elevated Alpha globulins (overestimates low albumin)  Can interfere with high hemoglobin count  Bromcresol Purple (BCP): an alternate dye, specific to albumin  Underestimates serum albumin in renal insufficiency  Bilirubin interference  HABA-2-(4-hydroxyazobenzene)-benzoic acid  Has low sensitivity but more specific for albumin  Interferences: salicylates, penicillin, conjugated bili rubin, sulfonamides  Biuret Method: looking at number of peptide bonds ; must remove globulins via salt precipitation first  Electrophoresis  Albumin Methods  Electrophoresis  Usually performed with abnormal total protein or albumin  When placed in an electric current, protein will move according to charge density, determined by pH of surrounding buffer  Cellulose acetate, agarose Plasma Proteins  Globulins  Proteins that consist of fractions or sections  These fractions consist of:  Alpha 1  Alpha 2  Beta  Gamma   GLOBULINS  1. Alpha 1-antitrypsin: a glycoprotein  inhibiting the protease neutrophil elastase; waste from phagocytosis  Minimize tissue damage Plasma   Increased in pregnancy or BC use An acute-phase reactant (increases with inflammation) Proteins  Major component of serum protein (90%)  Phenotype is MM (normal functional)  ZZ at risk for liver and lung disease; lack of alpha 1 antitrypsin  Decreased in Emphysema-associated pulmonary disease and severe juvenile hepatic disorders that may result in cirrhosis Plasma Proteins  Globulins  2. Alpha 1-Fetoprotein (AFP)  Levels peak at 13 weeks and decline at 34 weeks of pregnancy  Yolk sac (infants) liver (adults)  There should not be increased levels in adults  tumor marker , gonad tumors  INCREASED levels spina bifida, fetal distress and presence of twins  In normal babies it binds to hormone stadial  DECREASED risk for Down syndrome and trisomy 18 (Edward’s syndrome)  Screening 15-20 week gestation for pregnant women Plasma Proteins  Globulins  3. Alpha 1-Acid Glycoprotein: a major glycoprotein, negatively charged even in acid solutions.  5 carbohydrate unit attached to a polypeptide chain  Positive acute phase reactant  formation of membrane and fibers in collagen  DECREASED levels  nephrotic syndrome  INCREASED  following stress, inflammation, and tissue damage, acute myocardial infarction (AMI), trauma, pregnancy, cancer, pneumonia, rheumatoid arthritis, and surgery.  Useful diagnostic tool in neonates with bacterial infections Plasma Proteins  Globulins  4. Alpha 1-Antichymotrypsin: a member of the serine proteinase inhibitor (serpin) family  Inhibits enzymes cathepsin G, pancreatic elastase, mast cell chymase, and chymotrypsin  Deficiency  liver disease  Cleaves off peptide bonds; breaks down those enzymes  Migrates between alpha I and alpha II Plasma Proteins  Globulins  5. Inter-alpha-trypsin inhibitor: family of serine protease inhibitors  3 polypeptide subunits (one or two heavy chains + one light chain)  Acute phase reactant in inflammatory disease Plasma Proteins  Globulins  Haptoglobin: alpha glycoprotein synthesized in the hepatocytes  Consists of two alpha and two beta chains (tetramer)  Acute-phase reactant  Protein used to evaluate rheumatic disease, heart attacks, burns and necrotic symptoms  Increased levels hemolytic anemia  Binds free hemoglobin; keeps iron in our system Plasma Proteins  Globulins  Ceruloplasmin: copper-containing alpha-2 glycoprotein  90% of total serum copper; 10% is bound to albumin  INCREASED  in inflammation, severe infection, tissue damage, and with some cancers; also intake of oral estrogen and oral contraceptives  Acute phase reactant  DECREASED with Wilson’s disease, malnutrition, liver disease and necrotic syndrome  Wilsons disease: body store copper in different parts of body eg brain, liver  cirrhosis and neuro damage kaiser-Fleisher ring: copper in cornea Plasma Proteins  Globulins  Alpha 2-Macroglobulin: a large protein synthesized  Inhibit proteases  trypsin, pepsin, thrombin, kallikrein, and plasmin  INCREASED levels seen in nephrosis (10x more than normal), diabetes, and liver disease, pregnancy, contraceptive medications  DECREASED levels  acute pancreatitis  Major component of alpha 2 band in electrophoresis Plasma Proteins  Globulins  Transferrin (Siderophilin)(alpha 2): glycoprotein, a negative acute-phase protein synthesized primarily by the liver  Binds ferric iron when plasma is saturated  DECREASED levels  with infections, liver disease, malnutrition, and nephrotic syndrome  INCREASED levels iron deficiency anemia; free transferrin levels increase in pregnancy  Major component of beta band Plasma Proteins  Globulins  Lipoproteins: complexes of proteins and lipids whose function is to transport cholesterol, triglycerides, and phospholipids in the blood.  Subclasses: HDL, LDL, VLDL, Pre-beta Plasma Proteins  Globulins  Beta 2-Microglobulin: light chain component of the major histocompatibility complex  Found on surface of most nucleated cells (HLAs)  High concentrations present in lymphocytes  Small in size: filtered by the renal glomerulus and reabsorbed  Increased levels :Impaired kidney clearance, inflammation , rheumatoid arthritis and systemic lupus Plasma Proteins  Globulins  Complement: one of the natural defense mechanisms that protects the human body from infections  Proteins synthesized in the liver as single polypeptide chains  Increased levels in inflammation states  Decreased levels in malnutrition and hemolytic anemia Plasma Proteins  Globulins  Fibrinogen: one of the largest proteins in blood plasma, synthesized in liver and classified as glycoprotein  An acute-phase reactant  Increased levels seen in pregnancy, and use of oral contraceptives  Found in plasma  Concentration is proportional to clotting time  Decreased levels in coagulation diseases where fibrinogen is consumed Plasma Proteins  Globulins  C-Reactive Protein: a beta globulin synthesized in the liver and is one of the first acute-phase proteins to rise, non-specific monitor of inflammation  Used to monitor therapy, determine risk for cardio vascular disease  Elevated levels of CRP stimulate the production of tissue factor that initiates coagulation, activates complement, and binds to LDL in the atherosclerotic plaque  Increased levels seen in tissue necrosis, rheumatic fever, infections, myocardial infarction, rheumatoid arthritis, gout, viral infections, and carcinomatosis Plasma Proteins  Globulins  Immunoglobulins: Antibodies, are glycoproteins composed of 82% to 96% protein and 4% to 18% carbohydrate produced by B cells  There are 5 classes:  1.IgA  Increased in liver disease, various infections and autoimmune disorders  Decreased in disease of depressed protein synthesis  2. IgM: cannot cross placenta  First immunoglobulin to appear in response to antigen stimulation  Increased levels found in viral and bacterial diseases and Waldenstrom macroglobulinemia  Globulins  Immunoglobulins  3. IgG: can cross placenta and most abundant in blood plasma and lymph  Increased in liver disorders, infections and collagen disease  Decreased in presence of increased susceptibility to infection 4. IgE  Increased in allergies, asthma, hay fever, and during parasitic infections  5. IgD  Increased in liver disorders, connective tissue disorders and multiple myeloma Other Plasma Proteins of Importance  Myoglobin: single-chain globular protein of 153 amino acids, contains a heme prosthetic group  Used as a cardiac biomarker  Supplies oxygen to muscles  Toxic to kidneys  Detects rhabdomyolysis  destruction of muscle tissue (CK lvls) Other Plasma Proteins of Importance  Cardiac Troponin (cTn): represents a complex of regulatory proteins that are specific for heart muscle, can be detected up to 14 days  Troponin I (cTnI)  Troponin T (cTnT)  These are the “gold” standard in the diagnosis of acute coronary syndrome (ACS)  High sensitivity cardiac troponin (Hs-cTNT) is the latest generation that allows for detection of very low levels of troponin T BNP-Beta natriuretic peptide  Released by heart into the blood Congestive heart failure  Hearts muscles don’t pump enough blood, blood backs up into the lungs Fibronectin  fetal fibronectin (FFN)  Amniotic sac; keeps sac attached to lining uterus  fibronectin released when amniotic membrane disturbed  Detected via cervical fluid, detects pre-term labor  Tested week 22-34 of gestation Total Protein Abnormalities  1. Hypoproteinemia- not enough proteins in blood  Causes: excessive loss due to renal disease, liver disease, leakage into the GI tract in digestive system inflammation, blood loss in open wounds, internal bleeding, malnutrition, malabsorption, extensive burns, decreased intake  2. Hyperproteinemia- high protein concentration  dehydration and monoclonal disorders Methods of Analysis  Total nitrogen content  chemilumiescence  Measures all chemically bound nitrogen in the substance  Oxidized to nitric oxide, then mixed with ozone (O3) to form excited nitrogen dioxide molecule (NO2)  Molecule decays to ground state emitting chemiluminescent light  Total Protein  Plasma most often used to determine total protein  Interferences: Lipemia, hemolysis (falsely elevated; RBC release proteins)  Reference interval 6.5 to 8.3 g/dL (ambulatory) 6.0 to 7.8 g/dL (recumbent) Method for Analysis  Total Proteins  Biuret: most widely used  Cupric ions (CU2+) complex with groups involved in peptide bond in alkaline medium  Colormetric test to detect peptide bonds  The absorbance of the colored (purple complex) formed measured at 540 nm  Interferences: Lipemia, icterus, hemolysis  Dye Binding  Based on the ability of most proteins in serum to bind dyes  Causes a shift in the absorbance maximum of the dye from 465 to 595 nm  Higher absorbance the higher the concentration of analyte Methods of Analysis Total Proteins  Kjeldahl: quantitative (not used, tedious, time consuming)  Serum proteins precipitated with an organic acid (tungsten acid)  The nonprotein nitrogen is removed  Protein pellet digested in H2SO4 with heat  Potassium sulfate used o increase boiling point  The H2SO4 oxidizes the C, H, and S in protein to CO2, CO, H2O, and SO2  Nitrogen in protein is converted to ammonium bisulfite, measured by adding alkali and distilling ammonia into a standard boric acid solution  Ammonium borate formed is then titrated with HCL to determine amount of nitrogen in original protein solution Method for Analysis  Total Proteins  Electrophoresis: proteins separate based on electric charge  Serum  Major Serum proteins have a net negative charge at pH 8.6 and migrate toward the anode(+)  Albumin traveling the farthest, followed by alpha 1 globulins, alpha 2 globulins, beta globulins, and gamma globulins  The proteins are fixed in the medium, stained, and then quantified (densitometer)  Standard dyes Normal Pattern of Protein Electrophores is Proteins in Other Body Fluids  Urinary Protein  Qualitative test commonly performed using reagent test strip  Quantification performed on 24hr urine specimens  Elevated urine protein is an early indicator of renal disease  Increased in multiple myeloma, Waldenstrom macroglobulinemia  Microalbuminuria is an indicator of renal disease in diabetes mellitus(early indicator of diabetic nephrosis) Proteins in Other Body Fluids  CSF Protein  Accepted reference interval 15-45 mg/dL  Elevated levels may be caused by damage to the blood-brain barrier, during disease or following trauma  In conditions such as bacterial, viral, fungal meningitis, traumatic tap, multiple sclerosis, obstruction, neoplasm  Decreased in hyperthyroidism and when fluid is leaking from the CNS