3-Proteins

Zwitterion

no overall electrical charge but separate charges are (+) and (-) charge

Peptide Bond

Amide bond between carboxyl of one amino acid to amino group of another

levels of protein structure: PRIMARY

sequence ofAA

levels of protein structure: SECONDARY

specific organization of segments into alpha helix, beta sheet, beta turn

levels of protein structure: TERTIARY

folding into 3D structure

levels of protein structure: QUATERNARY

association of multiple peptide chains such as CK, LD, hemoglobin

Transamination

moves amino groups between compounds

Oxidative Deamination

-in liver and kidney

-liberation of alpha-ketoacids to enter pathway of energy

-liberation of ammonia to be used in urea cycle

Glucogenic vs ketogenic amino acids

What is total protein made of?

Albumin (60%) + globulins

Biuret reaction

-for serum total protein

-presence of ≥ 2 peptide bonds react with copper (Cu2+) in alkaline solution —→ purple complex

Kjeldahi

-not really used

-uses nitrogen content to measure protein

Albumin: Dye-binding methods

Bromecresol green (BCG)

Bromecresol purple (BCP)

Why can’t we use birute method for urine and CSF?

-too insensitive

-use Pryogallol red

What is coomassie brilliant blue used for?

immunoglobulins

Turbidimetric method

sulfosalicyclic or trichloroacetic acid precipitate proteins—> turbidity measured

Why is Pryogallol red the best?

-able to measure low amounts of proteins

How does Pyrogallol Red work?

-pyrogallol binds basic amino groups and forms a blue-purple complex

-measured at 600nm

-absorbance proportional to protein conc.

What ratios are useful for identifying multiple sclerosis?

-CSF IgG index: ratio of CSF to serum

-IgG synthesis rate: detects IgG chracteristic of MS

What must be done to urine before testing?

it must be concentrated since protein is so low

What does microalbumin measure?

low concentrations of albumin

What does microalbumin help monitor?

early-stage renal disease in diabetic patients

How often should microalbumin be measured in diabetic pts?

every 6 months

What causes Acute Phase Reactants (APR) to shift?

-Fever, and incr in granulocyte counts

-incr in hepatic synthesis of plasma proteins

-Loss in urine or fluid shifts

Positive vs Negative APRs

Positive= APR incr

-dilution or production incr

Negative= APR decr

-consumed or lost

List the Positive and Negative APRs

Which APR is the most sensitive/immediate?

C-reactive protein (CRP)

-may incr 1000x from baseline

How does hsCRP differ from CRP?

hsCRP uses more sample for better sensitivity to asses mild elevations

What is hsCRP used for?

hsCRP is used for Cardiac pts since it has a low sensitivity

At what pH is serum protein electrophoresis run?

8.6

What dyes are used in electrophoresis?

Amido black, ponceau S or Coomassie brilliant blue

For urine electrophoresis, why is it primarily done?

to detect free monoclonal IgG light chains or Bence-Jones (Multiple myeloma)

Electroendosmosis

proteins will form a negative ion cloud that moves to the anode BUT a positive ion cloud can form from the buffer used in electrophoresis and move toward the cathode

-since they are moving in opposite directions, it will create tension

What is the issue with Electroendosmosis

badly smears bands

Cathode is the ____charged electrode

(-)

Anode is the ____charged electrode

(+)

Which way will proteins migrate towards cathode or anode?

-Anode

-proteins have (-) due to carboxyl

Immunofixation Electrophpresis (IFE) process

1.set up sample and run electrophoresis

2.Overlay antisera in each channel

3.stain

4.locate comigrated fractions

What does immunofixation electrophoresis (IFE) help identify?

Monoclonal antibodies

Would you be able to identify which gammopathy with IFE?

Yes, using antisera after electrophoresis

PEP: Pre-albumin travels right before_____

albumin

Pre-albumin is used to indicate___.

protein nutrition

Albumin is the most abundant____.

serum protein (60%)

Albumin regulates____

blood colloidal osmotic pressure

What function does albumin do?

-retain fluid in vascular space

-transpot protein for things

• EX: like bili, H+, drugs, cortisol, fatty acids

What can lack of albumin lead to?

edema

Hyperalbuminemia is seen in

-sever dehydration #1

-fluid loss

-prolonged tourniquet

Hypoalbuminemia is seen in

-liver impairment

-edema or decr osmotic pressure

-renal loss/nephrotic syndrome

-malabsorption/malnutrition

-skin loss

-inflammation

Alpha1-antitrypsin is a __ ___ inhibitor.

serine protease (aka leukocyte elastase)

Deficiency in AAT leads to____ and _____

-emphysema: due to leukocyte elastase not being inhibited and cleaving more elastin

-liver disease

Where is Alpha1-Fetoprotein made?

fetal liver

Why is alpha1-fetoprotein tested?

-maternal screenings:

• incr = neural tube defects

• decr = down syndrome

-tumor marker for liver cancer

Alpha2-Macroglobulin is a major _____.

plasma proteinase inhibitor

Decr levels of alpha2-macroglobulin are seen in:

-pancreatitis

-carcinoma of the prostate

Incr levels of alpha2-macroglobulin are seen in:

-Nephrotic Syndrome

What does haptoglobin do?

transport free hgb

Incr levels of haptoglobin seen in:

-APP ( acute phase proteins)

-nephrotic syndrome

decr levels of haptoglobin seen in:

-hemolysis

-liver disease

-transfusion reactions

What is ceruloplasm?

-alpha2-globulin that contains 95% of ttl serum copper

-transports copper

incr levels of ceruloplasmin are seen in:

-acute phase reactant

-pregnancy

decr levels of ceruloplasmin are seen in:

Wilsons disease

What happens when someone has Wilsons disease?

-incr ttl body copper that deposits in tissues

Keyser Fleischer rings

-copper ring around eyes due to Wilsons disease

Transferrin

-beta globulin

-transports iron and lipoproteins

What disease does transferrin help diagnose?

anemia

Low levels of transferrin are seen in:

-impaired Hgb producition—>anemia

high levels of transferrin are seen in:

-IDA

-elevated lipoproteins (LDL)

What does beta2-microglobulin help asses?

renal tubular function

Incr beta2-microglobulin is seen in

-renal failure

-inflammation

-neoplasms

beta2-microglobulin can deposit as ____

amyloids

-leads to amyloidosis = dialysis

Where does fibrinogen migrate on electrophoresis

between beta and gamma

If a fibrinogen band is seen, what does it mean

incorrect sample collection since serum does not have fibrinogen

Decr in fibrinogen is seen in:

liver disease

Immunoglobulins are produced by ___

WBC (B+plasma cells)

Immunoglobulins are incr in:

-chronic inflammation

-cirrhosis or viral hepatitis

-collagen disease

-monoclonal paraproteins

-polyclonal gammopathy

Gammopathy: What is a para protein?

too much monoclonal Ig

-EX: multiple myeloma

How does para protein relate to multiple myeloma?

-produces monoclonal Ig

-cancer where plasma cells grow uncontrollably

What will be seen in the gamma region if someone has multiple myeloma?

M spike

What are Cryo-Globulins/ fibrinogens

proteins of IgM or IgG that ppt when less than 37 C

How would a pt with Cryo-Globulins/ fibrinogens present?

pain in extremities when cold (Reynauds phenomenon)

What is the common association with cryoglobulins?

Hep C

List alpha 1 proteins

-AAT

-AFP

List alpha 2 proteins

-alpha 2 macro

-haptoglobin

-ceruloplasmin

List beta proteins

-transferrin

-beta 2 microglobulin