Proteins and electrophoresis

What are some structural proteins?

Keratin, collagen, actin, myosin

What are some transport proteins?

Hemoglobin, transferring, ceruloplasmin, thyroid binding globulin 

What are some protein hormones?

Insulin, TSH, ACTH, PTH, GH

What is the most abundant protein in human plasma?

Albumin

What makes up globulins?

Alpha, beta and gamma 

What are fibrous proteins?

Collagen, elastins and keratins

Sort the proteins in order of lowest to highest isoelectric point.

Albumin<(alpha-2-macroglobun = beta I-lipoprotine=fibinogen)<transferrin<IgG

At what pH are most proteins’ pI?

Acidic

What happens to proteins when places in a buffer more alkaline than their pI?

They become negatively charged

What happens to proteins that are placed in a buffer more acidic than their pI?

They become more positively charged 

The anode is?

Positive

The cathode is?

Negative 

What do proteins separate based on?

Their pI and the pH of the buffer they are suspended in

What are the functions of albumin?

• maintaining fluid balance

• Carrier substance

• anti-oxidant activity

• buffer 

What is hyperalbuminemia associated with?

• Dehydration 

• Rare and of no clinical significance 

What is hypoalbuminemia associated with?

• Increased loss (nephrotic syndrome)

• Decreased production (nutritional deficit, liver failure)

What is bisalbuminemia?

• dimeric albumin

• considered normal 

What are some alpha-1-globulins?

• Alpha 1 antitrypsin

• alpha 1 glycoprotein (orosomucoid)

• alpha 1 fetoprotein (AFP)   

What is alpha 1 antitrypsin?

Protease inhibitor that binds to and inactivated trypsin

What is decreased alpha -1 antitrypsin associated with?

• pulmonary emphysema

• cirrhosis 

What is the only screening test for alpha 1 antitrypsin deficiency?

Serum protein electrophoresis (SPE)

What is alpha -acid glucoprotein?

An acute phase reactant 

What is alpha 1 fetoprotein?

• principal fetal protein, used to screen for fetal abnormalities

• Tumor marker (elevated in liver carcinoma)

What are some alpha 2 globulins?

• alpha 2 macroglobulin

• alpha 2 ceruloplasmin 

• alpha 2 haptoglobin 

What is alpha 2 macroglobulin?

• largest non-immunoglobulin in plasma

• protease inhibitor

When is alpha 2 macroglobulin increased?

Nephrotic syndrome

What is alpha 2 ceruloplasmin?

• Copper transport protein

• participates in plasma redox reaction 

What is Cu levels usual used to screen for?

Wilson’s disease

What is beta 1 transferrin?

Iron transport protein, also binds copper

When is transferrin increased?

In iron deficiency anemia, as well as pregnancy and estrogen therapy 

When is transferrin decreased?

Inflammation, malignancy or liver disease

When is beta 1 lipoprotein increased? Decreased?

• increased when LDL is elevated

• decreased in acute or chronic inflammation

What are the beta 1 globulins?

• transferrin

• Lipoprotein 

What are the beta 2 globulins?

• compliment proteins

• microglobulin 

What compliment proteins migrate in the beta region?

C3 and C4

When are compliment proteins decreased?

Decreased in genetic deficiencies and some autoimmune disorders that bind complement and increased in inflammation 

What is beta 2 microglobulin a measure of?

Renal tubular function, increased in renal failure

What is in the beta-gamma interzone?

• proteins that migrate between beta and gamma zones

  • C-reactive protein

  • IgA

  • fibrinogen 

What is in the gamma zone?

• Immunoglobulin and C-reactive proteins 

What is a single sharp peak in the gamma region associated with?

Monoclonal gammopathy

What is a broad increase in the gamma region associated with?

Inflammation, trauma, infection, etc

What does serum amyloid do?

• recruits immune cells to inflammatory site

• removes cholesterol from cholesterol-filled macrophages at injury site

When can urine protein be elevated that is not due to renal disease?

• exercise

• cold exposure

• stress

• orthostatic proteinuria 

What methods can be used to detect urine protein?

Flocculation methods like sulfosalicylic acid or trichoracetic acid 

What do elevations in CSF protein indicate?

• increased permeability of blood brain barrier

• increased synthesis of immunoglobulins 

What do oligoclonal bands in CSF indicate?

Multiple sclerosis 

What is true of the Biuret method of protein?

• uses alkaline CuSO4

• A minimum of 2 peptide bonds must be present to give a reaction 

What methods are used for albumin?

• Dye binding method

  • Bromocresol green

  • bromocresol purple 

What methods are used to separate proteins?

• chromatography

• gas-liquid

• high pressure liquid chromatography

• ion exchange column chromatography

• electrophoresis 

What factors influence the movement of molecules in electrophoresis?

• nature of the charged particle

• character of the buffer

• intensity of electric field

• time

• temperature

• voltage

• matrix of the medium

• endosmosis 

What is endomosis?

Large, highly charged protein may actually migrate toward the like-charged electrode. Influenced by movement of water toward the cathode 

What must be balanced for optimal electrophoresis results?

Speed and resolution

What does a higher voltage do?

Increases speed but heat causes evaporation of the buffer and may denature proteins

What can time cause?

Increased time increased separation but may cause diffusion of band

What does a higher ionic strength buffer cause?

Increased conductivity but enhances endosomatic effects

What do you get better separation with?

High ionic strength and less time 

What is seen in nephrotic syndrome?

• Loss of low molecular weigh protein like albumin and IgG

• retention of higher molecular weigh proteins (alpha-2-macroglobulin)

What is seen in polyclonal gammopathy?

Increase in gamma region

What is seen in liver disease?

• decreased albumin

• beta and gamma regions blurred (beta gamma bridge)

What pattern is seen in acute inflammation?

• albumin and gamma globin slightly decreased

• alpha 2 globulin very increased

What pattern is seen in alpha 1 anti trypsin deficiency?

Decrease in alpha 1 region 

What is seen in monoclonal gammopathy?

Large spike in gamma region

What is the minimum amount of urine needed for electrophoresis?

10 mL

What do most urine samples require?

• concentration

What is the pattern on alkaline hgb electrophoresis?

• O

• C

• S

• F

• A

What is the pattern on acidic hgb electrophorisis?

• F

• A

• S

• O

• C