Proteins (+ Electrophoresis Notes)

Proteins

Large biomolecules composed of amino acids linked through peptide bonds, essential for various biological functions including structure, enzymes, and signaling.

Functions of Proteins

• Carrier molecules

• Maintenance of osmotic pressure

• Immune response agents

• Enzymes

• Acts as a marker for nutrition

  • Albumin, Transferrin, Transthyretin or thyroxine binding prealbumin

• Acts as a marker for liver function

  • important in assessment of renal function

Essential Components of Proteins

Essential Components:

• Carbon - Forms the backbone of amino acids

• Hydrogen - Contributes to protein folding and stability

• Oxygen - Essential for peptide bonds and functional groups

• Sulfur - Forms disulfide bridges crucial for protein structure

• Nitrogen (16%) - Distinguishing feature from carbohydrates and lipids

Amino Acid Structure:

Each amino acid contains:

• An amino group (-NH₂)

• A carboxyl group (-COOH)

• A unique side chain (R group)

• A central carbon atom (α-carbon)

Amino Acid Analysis

Draw blood after a 6-8 hour fast in a heparin tube, removing plasma promptly. Avoid aspiration of platelet and white cell layers to prevent protein interference and hemolysis.

Protein Structure Hierarchy

1. Primary Structure:

The linear sequence of amino acids is joined by peptide bonds.

2. Secondary Structure:

Regular folding patterns stabilized by hydrogen bonds

3. Tertiary Structure:

Three-dimensional arrangement of the entire polypeptide chain.

4. Quaternary Structure:

Assembly of multiple polypeptide chains (subunits) into a functional protein complex.

Quaternary Structure of Protein Examples

• Hemoglobin (4 subunits: 2 α and 2 β chains)

• Immunoglobulins (4 chains: 2 heavy & 2 light)

Ampholyte (Zwitterion):

Amino acids at physiologic pH (7.4) exist as dipolar ions (both positive and negative charges on the same molecule) with

• Protonated amino group (-NH₃⁺)

• Deprotonated carboxyl group (- COO⁻)

Isoelectric Point (pI):

The pH at which a protein has no net electrical charge, important for

• Protein separation techniques

• Understanding protein behavior in solution

• Optimizing laboratory procedures

Protein Analysis

Sample: Serum, Plasma, CSF & Urine

• Refractometry: measure of refractive index due to solutes in a serum

  • rapid and simple

Albumin 

Major Serum Proteins: (3.5-5.0 g/dL)

• Maintains 80% plasma oncotic pressure

• Antioxidant activity

• pH buffering

• Transport protein for:

  • Fatty acids

  • Drugs

  • Bilirubin

  • Metals

  • Hormones

  • Vitamins

Albumin Methods: Dye: BCG and BCP

• BCG (bromcresol green): Albumin binds to dyes; causes shift in absorption maximum

  • More common

  • Sensitive - overestimates low albumin levels

• BCP (bromcresol purple): Albumin binds to dyes; causes shift in absorption maximum

Clinical Conditions Affecting Albumin:

• Hyperalbuminemia

• Hypoalbuminemia

• Analbuminemia

• Bisalbuminemia

Conditions + Albumin Level + Clinical Significance

• Hyperalbuminemia + Increase + Usually due to dehydration

• Hypoalbuminemia + Decrease + malnutrition, liver disease, nephrotic syndrome, protein losing enteropathy

• Analbuminemia + absent + rare genetic condition

• Bisalbuminemia + modified + presence of variant albumin

Oligoclonal Bands

Oligoclonal bands are distinct bands observed on electrophoresis, indicating the presence of immunoglobulins

• Darker band = higher conc,

Protein Fractions

• Prealbumin

• Albumin

• Alpha1 proteins

• Alpha 2 proteins

• Beta Proteins (Beta1 and Beta2)

• Gamma proteins

Acute Phase Proteins

• Positive APPs like C-reactive protein (CRP) and fibrinogen = increase rapidly

• Negative APPs such as albumin and transferrin decrease

• changes occur simultaneously

Acute Phase Proteins: Negative and Positive

• APP’s play a role in host defense, balane occurs due to compensation

  • Ex: Fibrinogen CRP, AAT, C3, AAG, a2-Macroglobulin, haptoglobin and ceruloplasmin

• APPs proteins increase, levels of negative APR proteins decrease

  • Ex: albumin, prealbumin and transferrin.

CRP: C-reactive proteins (beta)

first acute phase reactant to rise in response to inflammation and it is an independent cardiovascular risk factor, assessed using the hsCRP assay.

Alpha-1 Proteins

a class of serum proteins based on their electrophoretic mobility. Major examples include:

• α1-Antitrypsin (AAT): A protein that protects tissues from enzymes of inflammatory cells, particularly elastase

• α1-Fetoprotein (AFP): A protein produced by the fetal liver that has a function in binding and transporting nutrients.

α1-Antitrypsin (AAT)

Key protease inhibitor synthesized in the liver:

• protects tissues from neutrophil elastase

• Decreased in:

  • Emphysema

  • Liver cirrhosis

• Increased in:

  • Acute inflammation

  • Pregnancy

  • Oral contraceptive use

α1-Fetoprotein (AFP)

Important diagnostic marker: synthesized by fetal liver

• Major fetal protein in 2nd trimester

• Maternal serum screening for:

  • Neural tube defects (ONTD) - increased levels

  • Down syndrome - decreased levels

• Tumor marker for liver cancers:

  • Hepatocellular carcinoma

  • Germ cell tumors

Alpha-2 Proteins

• a2-Macroglobulin: Inhibits proteolytic enzymes, regulating proteolysis and protecting tissues.

• a2-Haptoglobin: Binds free hemoglobin, preventing kidney damage and aiding in immune response.

• a2-Ceruloplasmin: Carries copper, oxidizes Fe2+ to Fe3+ for iron transport, and provides antioxidant protection.

α2-Macroglobulin

Large alpha protein with multiple functions:

• Protease inhibition

  • trypsin, pepsin, and plasmin

• Hormone binding - insulin

• Increased in nephrotic syndrome - excessive protein loss in urine

How does a2-macroglobulin act as a hemoglobin binding protein?

• prevents hemoglobin loss in urine

• Decreased in hemolytic conditions

• increased in inflammatory states

Haptoglobin

• Binds free hemoglobin and transports it to the RE system for degradation, preventing renal loss of hemoglobin and iron.

• Levels decrease in hemolytic diseases (HDN and transfusion rxns)

• Increase in inflammation, burns, and nephrotic syndrome.

Ceruloplasmin

Copper-carrying protein:

• Decreased in Wilson's disease

• Increased in inflammation

• Important in iron metabolism

Beta Proteins: Transferrin and B2-Microglobulin

Transferrin: Iron transport protein

• Increased in iron deficiency

• Decreased in inflammation

• Negative acute phase reactant

B2-microglobulin: Important in renal function assessment:

• Filtered by glomerulus

• Reabsorbed by tubules

• Marker for tubular function

Fibrinogen

• analyzed in a green top tube (heparinized) while Fibrin clot formation observed in a red top tube (serum)

• Decreased levels: extensive coagulation (e.g., DIC), liver disease, or major blood loss

• Positive acute phase reactant

• Peaks between beta (β) and gamma (γ)

Immunoglobulins: Types + Structure + Function

• IgG + Monomer + Main antibody in secondary response

• IgM + Pentamer + First antibody in primary response

• IgA + Monomer/Dimer + Mucosal immunity

• IgD + Monomer + B-cell surface receptor

• IgE + Monomer + Allergic responses

Protein Electrophoresis: Fraction + Major Components + Clinical Significance

• Albumin + Albumin (3.5-5.0 g/dL) + Nutrition, liver function

• Alpha-1 + Acute phase reactions, liver disease

  • α1-Antitrypsin

  • α1-Acid glycoprotein

  • α1-Fetoprotein

• Alpha-2 + Nephrotic syndrome, acute inflammation

  • α2-Macroglobulin

  • Haptoglobin

  • Ceruloplasmin

• Beta + Iron status, complement activation

  • Transferrin

  • C3

  • β-Lipoprotein

• Gamma Immunoglobulins + Immune status + monoclonal gammopathies

Gamma globulins

• IgG, IgM, IgA, IgD and IgE

• composed of 2 identical heavy and 2 identical light (kappa and lambda) chains

• Single sharp peak in gamma region indicates monoclonal (coming from one cell line) gammopathy

• Increased in plasma cell malignancy (multiple myeloma), infection etc

Hypoproteinemia vs Hyperproteinemia

• Low protein levels due to renal loss, GI leakage, bleeding, and decreased intake from malnutrition or malabsorption

vs

• High protein levels caused by dehydration from vomiting/diarrhea and excessive production, such as Bence Jones protein in multiple myeloma.

Acute Phase Response:

• Decreased albumin

• Increased α1 and α2 globulins

• Elevated C-reactive protein

Multiple Myeloma:

• M-protein spike

• Sharp, narrow peak in gamma region

• Possible hypogammaglobulinemia

Chronic Liver Disease:

• Decreased albumin

• β-γ bridging

• Polyclonal gamma increase

Nephrotic Syndrome:

• Severe hypoalbuminemia

• Increased α2-macroglobulin

• Decreased gamma globulins

Advanced techniques/Testing Methods for specific protein analysis:

Method + Applications + Advantages

• Immunofixation + High specificity for paraprotein characterization

  • Monoclonal protein typing

  • Light chain identification

• Capillary Electrophoresis + High resolution, automation capability

  • Serum protein separation

  • Hemoglobin variants

• Mass Spectrometry + Precise molecular characterization

  •  Protein identification

  • Post-translational modifications

Positive Acute Phase Reactants

Protein + (Response Time) + Clinical Applications

• C-Reactive Protein: (6-8 hrs)

  • Bacterial infections

  • Inflammatory conditions

  • Cardiovascular risk

• Fribrinogen: (24-48 hrs)

  •  Coagulation status

  • Inflammation marker

Negative Acute Phase Reactants

Protein + Response + Clinical Significance

• Albumin + Decreased + Long-term nutritional status

• Transferrin + Decreased + Iron status assessment

• Prealbumin + Decreased + Short-term nutritional status

Electrophoresis example: Serum Proteins

When serum proteins are placed in a buffer of pH 8.6, they become (-) charged and migrate to an anode, while the buffer particles migrate to a cathode

What 2 properties affects the charge of Ampholytes in Buffer?

pH and Ionic Strength

• Neutral proteins do not migrate when pH = pI.

• If pH > pI, proteins carry a net negative charge and migrate to the anode (+ pole);

• if pH < pI, they carry a net positive charge and migrate to the cathode (-) pole.

• For example, at pH 8.6, albumin (pI 4.7) has a net negative charge and migrates to the anode.

Electrophoresis vs Electroendoosmosis

Electrophoresis involves the movement of charged particles in a fluid under the influence of an electric field, while electroendoosmosis refers to the movement of liquid in a porous medium due to an electric field, affecting the migration of particles.

Electroendoosmosis

the flow of buffer solution in contact with a negatively charged stationary surface, caused by an applied electric field, which attracts positive ions that drag surrounding water molecules towards the cathode.

Electroendoosmosis Results and Effects on Proteins

Weakly negatively charged proteins, such as gamma globulins, may be pushed toward the cathode due to buffer flow overriding the electrical pull, resulting in slower-than-expected migration. This tension can help achieve better separation of protein bands.

serum protein electrophoresis (SPE) Patterns

separates serum proteins into five main fractions: albumin, alpha-1 globulin, alpha-2 globulin, beta globulin, and gamma globulin

Patterns include: (A) Reference; (B) Monoclonal increase in γ area; (C) α1-Antitrypsin deficiency; (D) Nephrotic syndrome; (E) Inflammation; (F) Cirrhosis .

SPE: Reference Pattern

The normal densitometric pattern of serum protein electrophoresis, characterized by distinct albumin and globulin fractions, used as a baseline for comparison with other patterns.

SPE: Monoclonal Increase

A monoclonal spike in the gamma-globulin zone indicates a monoclonal gammopathy

SPE: a1-antitrypsin defiency

• reduced or absent alpha-1 globulin peak may indicate low AAT levels. 

• Low alpha-1 globulin levels may indicate AAT deficiency, which can lead to lung or liver disorders. 

SPE: Nephrotic Syndrome

• Decreased albumin and gamma globulins

• Increased alpha-2-globulins and beta-globulins

• Decreased total protein

SPE: Inflammation

Increased levels of alpha-1 and alpha-2 globulins, with possible elevation of immunoglobulins.

SPE: Cirrhosis

Decreased albumin levels and increased gamma globulins, often indicating liver dysfunction. Beta Bridge to gamma globulins present

SPEP: Hemolyzed Sample

Free hemoglobin will peak b/w a2 and b region while Hemoglobin-haptoglobin complexes will cause a peak in the a2 region

Proteins in Urine

• Elevated urine protein is an early indicator of renal impairment

• Bence Jones protein (Ig light chains) is present in multiple myeloma

Protein in cerebrospinal fluid (CSF)

Elevated gamma (IgG) levels produced in the CNS cause oligoclonal banding on high-resolution CSF electrophoresis, a marker for Multiple sclerosis

• CSF IgG also increases in bacterial, viral, and fungal meningitis.

oligoclonal bandings

are distinct bands of immunoglobulins seen in cerebrospinal fluid, indicating an autoimmune response or central nervous system pathology.

Immunofixation Electrophoresis (IFE)

identifying and characterizing monoclonal proteins (M-proteins) by separating them in a gel based on hevy and light chains.

• Forms dark visible bands that indicate monoclonal gammopathies

• Ex: multiple myeloma (dark band)

IgG vs IgM

IgG providing long-term immunity and is the main antibody in secondary response + most abundant while IgM is the first antibody produced during an immune response providing short-term immunity