Diagnostic Chemistry AA and Proteins

What are the fundamental building blocks of proteins/peptides?

Amino Acids

What roles do amino acids have?

1. Metabolism

2. Neurotransmission

3. Cell-to-cell signaling

Name the general functions of amino acids and their size

1. >40 AA

2. Provide intra-extracellular structure

-Catalysts (Enzymes)

-Contractility/motility mediation

-Facilitate molecular assembly

Name the general functions of peptides and their size

1. 2-5 AAs

2. Controls:

-Appetite

-Vascular tone

-Electrolyte balance

-Carb/ Mineral metabolism

Most AAs in proteins are alpha or beta?

Alpha

Except for glycine (the simplest), how many ionizable sites do alpha AAs contain?

2

Describe the general structure of Amino Acids

1. Amino Group (NH2) (proton-accepting)

2. Carboxyl group (COOH), acidic group (sulfonate [SO3]), or carboxylate (COO). (Proton-donating)

3. Hydrogen

4. R Group (makes each AA different)

Most AAs are which enantiomer (mirror image) configuration?

L

Define Essential amino acids

AAs that are not produced by humans but are needed to be consumed through diet

Acid-Base properties depend on the attached?

1. Amino Acid Group

2. Carboxyl Group

3. Basic/Acidic group in the side chain

True or false: At different pH levels AAs can gain or lose protons (Net Charge)

True

Define Amphoteric behavior

Charge depends on pH

-The Cation to Zwitterion occurs at pK1

-The Zwitterion to anion change occurs at pK2

At Low pH there are more H+ ions therefore what charge is the AA?

Cation (positive)

-The amino group (NH3+) is protanated

-Carboxyl stays COOH

At neutral pH what is the charge of AA?

Neutral Charge (Both charges present= 0)

-As pH rises, carboxyl group looses an H (COO)

-NH3+ keeps its charge

-Known as a Zwitterion or Ampholyte

-Most common form a physiological pH of 7.4

At High pH what is the charge of AA?

Anion (Negative charge)

-The amino group looses it H (NH2)

-pH can affect AA behavior and function

Definition of Isoelectric Point (PI)

PI is the pH which the molecule carries no net charge.

-It exists as a zwitterion and will not migrate in an electric field

What is the formula for PI?

PI= 1/2 (Pk1 + Pk2)

At a given pH the net charge determines a proteins?

-Solubility

-Structure

-Interactions

Define a peptide bond

The bond that links amino acids together; formed by a dehydration reaction

The C-N bond is what type of bond?

Amine bond

-N-Terminal residue = amino group free

-C-Terminal residue = carboxyl group is free

Where is the site of synthesis for serum proteins

1. Ribosomes of hepatocytes

2. Ribosomes of plasma cells

Describe the primary structure of a protein

-Linear sequence of alpha amino acids

-Increased diversity in proteins due to post translational modifications of AAs

-Help by Peptide bonds

Describe the secondary structure of protein

The peptide backbone can exist in three possible structures:

1. Alpha helix

-Most common structure

-Coiled backbone (random)

2. Beta sheet (fold)

-Rigid, stable core of a protein (most stable)

3. Beta turn

-Flexible connecting hinge that enables polypeptide chain folding

*** Stabilized by disulfide bonds and hydrogen bonds

Describe the tertiary structure of protein

-Refers to how polypeptide chains fold and form a compact 3D structure

-Protein function is dictated by this

-Stabilized by hydrogen bonds, van der Waals forces, and hydrophobic interactions

Describe the quaternary structure of a protein

-Incorporation of multiple polypeptide chains or subunits into a larger aggregate unit

-Can be simple or complex

-subunit can be identical or different

Define oligopeptide

< 5 amino acids

Define polypeptide

6-30 Amino acids

Define Simple proteins

Only has AAs

Define Conjugated proteins

-Contain nonprotein groups

-When non protein groups goes missing it become apoproteins

-EX: lipoprotein - lipids = apolipoprotein

Define Metalloproteins

Proteins with a strongly bound metal ion (or complex metals)

Define Lipoprotein

Protein that contains lipids

-contains cholesterol, triglycerides or phospholipids

Define Glycoprotein

Simple protein and carbohydrate (covalently linked carbs)

-Carbs make up less than 4% of toal weight

Define Mucoprotein

Complex carbs covalently linked to apoprotein

- Make up More than 4% of total weight

Define Nucleoproteins

-Proteins with a Nucleic acid prosthetic group (RNA/DNA)

Define Globular proteins

Soluble, compact, folded, coiled chains, symmetric

-Contain hydrophobic cores

-ALBUMINS

Define Fibrous proteins

Insoluble in water and asymmetric

-Highly resistant to proteolytic enzymes

-EX: Fibrinogen, Hair, keratin, collagen

List all the physical properties of proteins and their lab relevance

1. Absorption

-Diversity in physical structures or physical properties

-Tyrosine, tryptophan and phenylalanine absorb at 280 nm of light

-Oxygenated hemoglobin absorbs at 540-570 nm

-Provides an estimate of protein concentration in solution

2. Differential Solubility

-Different proteins dissolve to different degrees in a solvent

-Affected by pH, ionic strength, temperature, and dielectric constant of the solvent

-Affects net charge

-Proteins may be separated in solution

3. Molecular size

-Physical 3D space occupied by molecule

-Demonstrated by differential migration through molecular filters (electrophoresis)

4. Molecular Mass

-Sum of all atomic masses of all atoms in a molecule

-Laser desorption (MALDI-TOF) measures mass-to-charge ratio after ionizing molecules and observes how long they take to reach a detector

5. Electrical Charge

-Measured by ion-exchange chromatography and electrophoresis by separating proteins based on charge

6. Surface Adsorption

-Proteins can be separated based on their affinity for a variety of physical surfaces

-Reverse-phase chromatograph exploits interactions of hydrophobic molecular moieties and hydrophobic surfaces

7. Affinity chromatography

-Based on specific binding affinity related to size or charge

-Target protein of interest binds to beads coated in ligands; proteins not of interest wash away

Define Denaturation

Structural peptide bonds broken but primary structure remains intact (exposure to heat, chemicals or UV)

Define Hydrolysis

Destruction of primary structure (Exposure to acid or digestive enzymes)

Whats the main difference between denaturation and hydrolysis

Denaturation changes protein shape, and hydrolysis destroys protein backbone

*** Both result in loss of function

What is the flow of metabolism through the body?

Starts in the GI tract

-HCL and pepsin denature protein

*AAs are absorbed in the jejunum and goes into portal circulations and AA pools

*AAs remain in the pools until liver and other organs use them for protein synthesis

-Important with respect to essential AAs

Where are most plasma proteins synthesized?

The Liver

Describe Protein synthesis:

1. Transcription

-Initiation: RNA polymerase unwinds DNA and synthesizes RNA at transcription site

-Elongation: ribonucleotides are added that complement the DNA strand sequence

-Termination: Signal dependent= releases mRNA transcript

2. mRNA processing

-5 prime cap added

-Splicing

-3 prime poly A tail added to prevent degradation and regulate translation

3. Translation

-Ribosomes assemble at start codon

-Ribosomes incorporate amino acids via codons and tRNA anticodons

- Peptide release triggered by stop codon

List the main Functions of proteins?

1. Colloidal Osmotic Pressure

-Maintains water distribution

2. Structural support

-Collagen: Fibrous protein in connective tissue (25% of total body weight)

-Keratin: Hair and nails

3. Transport molecules

-Binds with ions or molecules to transport them to another site in body for storage or conjugation

-Albumin binds bilirubin for transport to liver

-Transferrin binds iron for transport to tissues

4. Enzymes

-Catalyze reactions important in digestion, defense, breathing, muscle, nerve

5. Peptide hormones

-insulin

6. Coagulation factors

-Maintains hemostasis

7. Hemoglobin

-Oxygen transport

8. Antibodies

An increase in protein in plasma means?

A decrease in H2O in interstitial space

A decrease in protein in plasma means?

An increase in H2O in interstitial space

Define Hyperproteinemia:

Positive Nitrogen Balance

-Dietary nitrogen intake is greater than excretion or loss of nitrogen (in urine)

Define Hypoproteinemia:

Negative Nitrogen Balance

-Excretion of nitrogen is greater than intake or synthesis of protein

What are the categories of causes of Hyperproteinemia?

1. Dehydration

-Low water intake

-Salt-losing syndromes

-Addison's disease

-DKA

2. Increase in globulins

-Multiple myeloma

-Waldenstroms's macroglobulinemia

-HIV

What are the categories of causes of Hypoproteinemia?

1. Increase in plasma water volume

-IV infusions

-Water intoxications

2. Increase in protein loss

-blood loss

-burn patients

3. Decreased intake

-Malnutrition

4. Decreased synthesis

-Liver disease

What is the most common method for total protein determination?

Biuret method

What is the principle of Biuret method?

-Based on presence of peptide bonds in all proteins

-Serum/plasma is treated with copper ions to form a colored complex

-Absorbs light at 540 nm

-The higher the color intensity (more purple) = proportional to number of peptide bonds Increase total protein

What are some interferences that occur with the Biuret test?

1. Hemolysis

-False +

2. Lipemia

- False +

3. Icteric (Lots of bilirubin)

- False -

4. Turbidity

- False +

What is the principle of Protein analysis Refractometry testing?

Rapid but approximate

-Based on light refraction

-Plasma protein solute diluted in water

=If increase in protein =Increase of bending of light

-Nonportein compounds in serum don't significantly impact refractive index

What is the principle of protein analysis of Turbidimetric assays?

1. Used in fluids with low protein content (urine)

2. Based on decrease in light transmission caused by particle formation

-Amount of light scattered depends on concentration and particle size

3. Performed with photodetectors or spectrophotometers (sensitive)

What are some interferences for Turbidimetric assays?

1. Lipemia

2. Bilirubin

3. Paraproteins

4. Particulates

What is the main difference between nephelometry and turbidimetry test?

1. Nephelometry measures scattered light

2. Turbidmetry measures transmitted light

What is the principle of Nephelometry?

1. Measurement of light scattered by particles in solution (15-90 degrees)

2. Scatter depends on light wavelength and particle size

3. Antibodies targeting proteins often used to make large antigen-antibody complexes which increases sensitivity of measurement

4. NOT used for total protein in serum

5. Used to measure specific proteins

-Can be used to measure total protein in CSF or Urine

Describe the principle of Immunochemical methods:

1. Enzyme immunoassays (see immune think antigen-antibody)

-ELISA

-CLIA

2. Used to measure a variety of individual proteins

3. STEPS

-Capture antibody coats sample well: protein antigen of interest in sample binds to antibody to form ag-ab complex

-2nd detection antibody added to bind complex then enzyme reagent added

-Substrate added to mixture and reacts with enzyme reagent to produce color or fluorescence change

-Intensity measured and compared with a standard curve to determine concentration

What are the interferences for immunochemical methods?

1. Heterophile antibodies (weak/nonspecific)

2. Hemolysis, lipemia

3. Cross-reactivity with similar molecules

4. Hook effect

5. Matrix effects (lipid or pH interference)

6. Poor washing

7. presence of enzyme inhibitors

What is the principle of protein analysis Dye-binding Test?

1. Common in research/teaching

2. Negatively-charged dye (Coumassie Blue) is dissolved in acid and binds to positively-charged proteins

-Before binding = red/brown 465nm

-After binding = blue 595nm

-Shift in color = increase in protein

What is the most common albumin dye-binding method?

Bromcresol Green

What is the most specific albumin dye-binding method?

Bromcresol Purple

-No significant globulin binding

What albumin dye-binding method is no longer used?

Methyl orange

What are the main interferences for dye-binding methods?

Chromogens: bilirubin and hemoglobin

What is the principle for urinary protein analysis?

1. Protein error of indicators (most common)

-Indicator: tetrabromophenol blue = yellow when no protein

-Color changes as protein accepts ions

2. Semiquantitative

-Roughly correlates with protein concentration (mg/dL)

-1+, 2+, 3+, 4+

3. Strips more sensitive to albumins than globulins

4. Correlate results with urine specific gravity

-If protein + in dilute urine is is much more significant than darker urine

In urinary protein how does Sulfosalicylic acid (SSA) work?

1. Type of turbidimetric assay for total protein

2. Protein precipitates after addition of SSA

3. Increases turbidimetry = Increase protein

-NOT common

What substances can precipitate in turbidemtric urinary protein analysis causing false positives?

1. X-ray dyes

2. Antibiotics

3. Tolbutamide metabolites

What is the principle of Protein Electrophoresis?

Migration of charged particles in a liquid medium within an electrical field

-Proteins can carry both positive and negative charges

What are the two electrodes in protein electrophoresis?

Anode (positive)

Cathode (negative)

What type of gel is electrophoresis performed on?

Agarose in an alkaline buffer (pH 8.6)

-Most serum proteins have net negative so move to anode

Do you use serum or plasma for electrophoresis?

SERUM

-plasma contains fibrinogen

Which factors affect migration of protein electrophoresis?

1. Buffer pH

2. pI of each protein

3. Molecule size/shape

4. Electrical field strength

5. temp

The larger/darker the band in electrophoresis?

The greater the protein concentrations

What are the five peaks in electrophoresis?

1. Albumin

2. alpha1

3. alpha2

4.Beta

5. Gamma

What is the electrophoresis sign for active cirrhosis?

Beta Gamma Bridging

What is the electrophoresis sign for Nephrotic Syndrome?

A decrease in albumin and Gamma bands

An increase in Alpha2 bands

What is Selective proteinuria?

Smaller proteins (albumins and Gamma) are excreted while larger A2 is retained

-Albumin must decrease by 1/3 to be noticed on electrophoresis

What is the electrophoresis sign of Monoclonal gammopathy?

Increased Gamma band

Most common cause of Monoclonal gammothapy?

MGUS a benign condition with age, genetics and immune system

-Associated with multiple myeloma and Waldenstrom macroglobulinemia

What is the electrophoresis sign for Polyclonal gammopathy?

Decrease in gamma band (elevation peak) Beta has no complete fusion

What is polyclonal gammothapy seen in?

Chronic liver disease

Chronic inflammatory disease

What is the electrophoresis pattern for Acute Phase Reactions?

Increase in both a1 and a2 bands

-Increase in b-globulins C3, C4, and c-reactive proteins and decrease in albumin and transferrin

What is seen with acute phase reactions?

Cancer, RA, hepatitis, trauma, burns