4. Proteins, Measurement, Electrophoresis Flashcards
Proteins & Amino Acids
- 20% of the human body is made up of proteins
- Proteins are large, complex molecules that are critical for normal functioning cells
- Proteins are essential for the structure, function, and regulation of the body’s tissues and organs.
- Proteins are made up of smaller units called amino acids, which are building blocks of proteins. They are attached to one another by peptide bonds forming a long chain of proteins
- Amino acids are compounds having both a carboxyl group (-COOH) and an amino group (-NH2)
Amino acid reactions are..
highly pH dependent
pH Dependent Properties
Zwitterionic* structures contain both N-H+ and COO-.
• At low pH, protonate COO-.
• At higher pH : lose H on N
• Isoelectric** pH: differs for each amino acid (due to structural differences)
Zwitterion: Definition
A molecule or ion having separate positively and negatively charged groups
Isoelectric point: Definition
The pH of a solution at which the net charge of a protein becomes zero
Leucine ionic forms
- Cation below pH 2.4
- Neutral between pH 2.4 and 9.6
- Anionic above pH 9.6
Dipeptides
Consider the 2 amino acids glycine (G) and alanine (A).
How many dipeptides can be made if these are randomly mixed?
GG, AA, GA and AG
N terminal on LHS; C terminal on RHS
Tripeptides
Consider amino acids Glycine (G), Alanine (A) and Phenylalanine (P)
How many different tripeptides are possible if each amino acid must be present?
Possible Tripeptides
- 3 choices for the N-terminal amino acid
- 2 choices for middle
- 1 choice for the C terminal amino acid
- Thus 3 x2 x1 =6 choices if each aa must be present.
- But total number possible is 3 x3x3 =27; includes AAA, PPP, GGG, etc.
How can you determine the overall protein structure of a molecule?
Sequence of amino acids can be determined using the enzyme carboxypeptidase (cleaves one aa at a time from the C terminal end)
Levels of Protein Structure
(1) Primary structure - the sequence of amino acids in the peptide chain and the location of the disulfide bridges
(2) Secondary structure - a description of the conformation/ shape of the backbone of the protein
(3) Tertiary structure - a description of the 3D structure of the entire polypeptide
* * If the protein has more than one chain, it can have a quaternary structure
Secondary Structure of Proteins
- Is the fixed arrangement of amino acids resulting from interactions between amide linkages that are close to each other in the protein chain
- Interactions can be hydrogen bonds (~ 5 kcal/mol each)
- Many H bonds are sufficient to define the shape
Ionic Interactions in Proteins
- “salt bridges”
- Involve COO- and remote NH3+ groups
- Along with H bonding and dispersion forces, these are responsible for the overall shape or “conformation” of the protein
Tertiary Structure of Proteins
- Arises from weaker attractive forces (non polar dispersion forces) between hydrophobic parts of the same chain that are widely separated in the primary structure, but close in space
- “intramolecular”
- Results in chain twisting and folding
Tertiary Structure of Protein: Braids
- Collagen-a fibrous protein (precursor of gelatin) has a triple helix structure-some elasticity due to interchain interactions
- Hemoglobin (a globular protein)
Hemoglobin (H)
H has 4 polypeptide chains : carries O2, CO2 and H+ in the blood, and possesses quaternary structure; Fe II containing heme unit in each chain that binds O2
- Hemoglobin has 4 polypeptide chains and possesses quaternary structure
Myoglobin (M)
M has a single chain of 153 amino acids: carries O2 from the blood vessels to the muscles and stores it until needed; Fe II containing heme unit in each chain that binds O2
- Myoglobin cannot have quaternary structure since it has only one polypeptide chain
Enzyme structure
- Many enzymes are proteins and their specific binding properties to a substrate depend on their overall molecular shape or “conformation”
- Lock and key mechanism for activity
Denaturation
- Any physical or chemical process that changes the protein structure and makes it incapable of performing its normal function
- Whether denaturation is reversible depends on the protein and the extent of denaturation
e. g. heating egg whites (irreversible) · ‘permanent’ waving of hair (reversible)
Contractile proteins
muscle
Hormones
insulin, growth hormone
Neurotransmitters
endorphins
Storage proteins
store nutrients
e.g. seeds, case in milk
Transport proteins
hemoglobin
Structural proteins
collagen, keratins
Protective proteins
antibodies
Toxins
snake venom, botulinum
Protein – Daily Requirements
- Average adult contains ~10kg of protein; ~300g is replaced daily by recycling and intake
- We need to take in ~70g of high quality protein or ~80g of lower quality
- This varies with age, size and energy demand,
eg. infants: 1.8g/kg/day
children: 1.0g/kg/day
adults: 0.8g/kg/day
Recommended: ~15% of daily Caloric intake
Since proteins are a major source of nitrogen, they are constantly being broken down and reconstructed. Protein is lost in:
urine, fecal material, sweat, hair/nail cuttings, and sloughed skin
Essential Amino Acids
The essential amino acids (10) are those that our bodies cannot synthesize. We must obtain them from our dietary intake
e.g. histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine (and arginine in infants)
Nonessential Amino Acids
The non-essential a.a.(10) can be synthesized in our bodies from breakdown products of metabolism
Mechanical Agitation
Beating egg whites-proteins denature at the surface of the air bubbles
Cream of tartar (the dipotassium salt of tartaric acid) is added to beaten egg whites to keep them stiff for mousse and meringue preparation, by raising the pH
Disinfection by Denaturation
Ethanol acts via denaturation of bacterial protein
Detergents (and soaps) disrupt association of protein sidechains of bacterial protein
Vegetarian Diets
The main challenge is to get enough high-quality protein with the correct balance of essential amino acids
Complete or High-Quality protein
Contains all the essential amino acids in about the same ratio as they occur in human protein
eg. meat, fish, poultry
Incomplete or Low-Quality Protein
Is deficient in one or more of the essential amino acids
eg. protein from plant sources
Complementary Proteins
Combinations of incomplete or low-quality proteins that taken together provide about the same ratio of essential amino acids as do high-quality proteins
e.g. Most of the people of the world depend on grains, not meat, as their major source of proteins. Many of these people have developed food combinations containing complementary proteins that allow them to live without suffering from malnutrition
Protein Determination
The key roles which plasma proteins play in bodily function, together with the relative ease of assaying them, makes their determination a valuable diagnostic tool as well as a way to monitor clinical progress
Variations in plasma protein concentrations can be due to any of three changes:
(1) in the rate of protein synthesis,
(2) the rate of removal, and
(3) in the volume of distribution
In spite of functional differences between the various serum proteins, they have certain common biophysical and biochemical properties. These include:
(1) a basic composition of carbon, hydrogen, nitrogen and oxygen;
(2) a backbone of covalent peptide bonds which join the amino acid units together
(3) absorption in the ultraviolet region
** Based on these properties, laboratory methods have been developed to determine the concentration of proteins in serum often with the assumption that each of the several hundred individual proteins present in serum reacts similarly in chemical reactions
Serum Total Protein (Plasma total protein or total protein)
A biochemical test for measuring the total amount of protein in blood plasma or serum
Protein in the plasma is made up of..
Albumin and Globulins; the globulin in turn is made up of α1, α2, β, and γ globulins
These fractions can be quantitated using protein electrophoresis, but the total protein test is a faster and cheaper test that estimates the total of all fractions together
The traditional method for measuring total protein uses the biuret reagent, but other chemical methods are also available
Protein Determination: Methods
Method 1: Kjeldahl; quantitative, protein nitrogen determination
Method 2: Absorption; quantitative, absorption at 210 nm (?)
Method 3: refractometry; quantitative
Method 4: Biuret; quantitative, increased absorption at 540 nm (?)
Protein Determination: Specimens
Serum or plasma may be used, and usually yield comparable results, though, because of the presence of fibrinogen
Plasma protein is about 0.3–0.5 g/L greater than serum protein
Fast Specimen – Importance
- A fasting specimen is not required but may be desirable to decrease lipemia
- Total protein is stable in serum and plasma for
- 1 week at room temperature and at least 60 days at –20°C
Specimens: The concentration of proteins in plasma is affected by..
POSTURE – : an increase in concentration of 10-20% occurs within 30 minutes of becoming upright after a period of recumbency
What happens when you apply a tourniquet before an acupuncture?
If a tourniquet is applied before venipuncture, a significant rise in protein concentration can occur within a few minutes
The change in protein concentration is caused by increased diffusion of fluid from the vascular into the interstitial compartment
These effects must be borne in mind when blood is being drawn for the determination of protein concentration
Hypoproteinemia
- Excessive loss as in renal disease,
- GI leakage,
- Excessive bleeding,
- Severe burns
- Excessive catabolism
- Liver disease
Hyperproteinuria
- Dehydration
- Monoclonal increase
- Polyclonal increase
(Only disorders affecting the concentration of albumin and/or the immunoglobulins will give rise to abnormal total protein levels. Other serum proteins are rarely present in high enough concentrations for changes to have a significant overall effect)
Biuret Method
The Biuret reagent is made of (NaOH) and copper (II) sulfate (CuSO4), together with potassium sodium tartrate (KNaC4H4O6·4H2O)
A blue reagent which turns violet in the presence of proteins, the Sodium hydroxide does not participate in the reaction at all, but is merely there to provide an alkaline medium so that the reaction can take place
Biuret Method: Principle
Peptide bonds of proteins react with tartrate-complexed cupric ions in alkaline solutions to form a colored product.
In a positive test, a copper(II) ion is reduced to copper(I), which forms a complex with the nitrogen and carbon of the peptide bonds in an alkaline solution
A violet color indicates the presence of proteins.
It is possible to use the Biuret reaction to determine the concentration of proteins because (for most proteins) peptide bonds occur with approximately the same frequency per gram of material.The intensity of the color, and hence the absorption at 540 nm, is directly proportional to the protein concentration, and can be determined spectrophotometrically at 540 nm
Reference Range for Total Proteins
Total protein -6.4-8.3 g/dL or 64-83 g/L
Albumin-3.5-5 g/dL or 35-50 g/L
Globulin -2.3-3.4 g/dL
Alpha (α)-1 globulin -0.1-0.3 g/dL or 1-3 g/L
Alpha (α)-2 globulin-0.6-1 g/dL or 6-10 g/L
Beta (β) globulin-0.7-1.1 g/dL or 7-11 g/L
Albumin: Definition
The most abundant circulating plasma protein (40–60 % of the total)
Albumin: Roles
- the maintenance of the colloid osmotic pressure of the blood,
- in transport of various ions, acids, and hormones.
- It is a globular protein with a molecular weight of approximately 66,000 Da and is unique among major plasma proteins in containing no carbohydrate
- It has a relatively low content of tryptophan and is an anion at pH 7.4
These properties have been exploited in the estimation of albumin in body fluids
Albumin Method 1: Precipitation (quantitative)
Salt fractionation, Solvent fractionation, Acid fractionation
Principle of analysis: Changes of net charge of protein result in precipitation
Albumin Method 2: Tryptophan content (quantitative)
Principle of analysis: Glyoxylic acid + tryptophan in globulin Purple chromogen (read at 540 nm); Total protein – globulin = albumin
Albumin Method 3: Electrophoresis (quantitative)
Principle of analysis: Albumin is separated from other proteins in electrical field; percent staining of albumin fraction multiplied by total protein value
Albumin Method 4: Immunochemical
Radial immunodiffusion; Turbidimetry; Nephelometry; Radioimmunoassay; Enzyme immunoassay
Albumin Method 5: Dye Binding (quantitative)
Methyl orange; BCG (bromcresol green); BCP (bromcresol purple)
Albumin Method 6: Dye Binding (semiquantitative)
Bromphenol blue in test strip changes color from yellow to blue in presence of albumin; most commonly used test for urine protein
Albumin: Specimen
Serum is the specimen of choice
Fasting is not required, although it may be desirable since marked lipemia interferes in the BCG assay
Venostasis should be avoided when collecting samples since hemoconcentration increases the apparent concentrations of albumin and other plasma proteins
Albumin: Reference Interval
3.4 to 5.4 g/dL (34 to 54 g/L)
Albumin: Clinical Significance
Plasma albumin levels, although important for management and follow-up, have very little value in clinical diagnosis
Hyperalbuminemia is usually attributable to:
dehydration or hemoconcentration.
Hypoalbuminemia is usually the result of
hemodilution, a rate of synthesis less than the albumin loss,
diseases that cause a large albumin loss from urine, skin, or intestine, and increased catabolism observed in fevers, untreated diabetes mellitus, and hyperthyroidism
Albumin: Dye-Binding Techniques
Serum albumin is most often assayed using dye-binding techniques
Albumin preferentially binds to anionic dyes that do not attract globulins
Bromcresol purple (BCP) and bromcresol green (BCG) are most commonly used
The amount of light absorbed by the albumin–dye complex is proportional to the amount of albumin present
Albumin: Bromcesol Green (BCG) Complexes
BCG + Albumin BCG-albumin complex (Bromcresol Green)
PRINCIPLE
BCG complexes with albumin, resulting in the dye having a spectral shift
The presence of albumin increases the absorbance at 546 nm
At pH 4.3, BCG + Albumin BCG-albumin complex
Albumin: Bromocresol Purple (BCP) Complexes
BCP + Albumin BCG-albumin complex (Bromocresol Purple)
Principle
BCP complexes with albumin, resulting in the dye having a spectral shift
The presence of albumin increases the absorbance at 610 nm
BCP + Albumin BCP-albumin complex
Folin-Ciocalteu (Lowry) Assay
- Sensitive over a wide range
- Can be performed at room temperature
- 10-20 times more sensitive than UV detection
- Can be performed in a microplate format
- Many substances interfere with the assay (e.g. strong acids, ammonium sulfate)
- Takes a considerable amount of time to perform
- The assay is photosensitive
Bicinchoninic Acid (BCA) Assay
- Very sensitive and rapid if you use elevated temperatures
- Compatible with many detergents
- Working reagent is stable
- Little variation in response between different proteins
- Broad linear working range
- Reaction does not go to completion
Dye-Binding (Bradford) Assay
- CBBG primarily responds to arginine residues
- If you have a rich protein, you may need to find a standard that is arginine rich as well
- CBBG binds to these residues in the anionic form
- Absorbance max is 595 nm
- Fast and inexpensive
- Highly specific for protein
- Compatible with a wide range of substances
- Dye reagent is complex (stable for approximately one hour)
- Response to different proteins can vary widely
