Laboratory Analysis of Proteins – Review Flashcards

34 question-and-answer flashcards covering reference ranges, analytical methods (Kjeldahl, Biuret, dye-binding, turbidimetry), specimen issues, albumin and globulin assays, UV absorbance, electrophoresis principles, SPE, IFE, capillary electrophoresis, isoelectric focusing, and clinical interpretations.

What is the adult reference range for total serum protein?

6.0–8.0 g/dL

What is the adult reference range for serum albumin?

3.5–5.2 g/dL

How is serum globulin concentration usually calculated?

Globulins = Total protein − Albumin

What two assumptions underlie empirical total-protein methods?

(1) All proteins contain 16 % nitrogen; (2) All proteins react chemically like every other protein.

Why is hemolysis unacceptable for total-protein measurement?

RBC lysis releases intracellular proteins into serum, falsely elevating the result.

By what age do total serum protein values reach adult levels?

Approximately 3 years of age.

Briefly describe the principle of the Kjeldahl method for protein determination.

Protein nitrogen is converted to ammonium, distilled, and titrated; the measured nitrogen is converted to total protein assuming 16 % N content.

What formula converts Kjeldahl nitrogen to total protein?

Total Protein (g) = Measured nitrogen / 0.16

Give two reasons the Kjeldahl method is rarely used clinically today.

It is cumbersome/time-consuming and relies on the assumption of uniform 16 % nitrogen content.

What physical phenomenon is measured in turbidimetry and nephelometry for proteins?

Light scattering by fine protein precipitates.

For which fluids are turbidimetric/nephelometric protein assays most common?

Urine, cerebrospinal fluid (CSF), and for IgG or complement in serum.

Why is turbidimetry considered less accurate than other total-protein methods?

It is difficult to control precipitate particle size, leading to variable light scattering.

State the chemical basis of the Biuret reaction for total protein.

In alkaline solution, cupric ions (Cu²⁺) complex with peptide bonds, forming a violet chelate.

At what wavelength is the Biuret complex typically read?

≈ 540 nm

Which specimen interference can invalidate Biuret absorbance readings?

Lipemia (excess lipids).

What main limitation affects dye-binding methods for total serum proteins?

Individual proteins bind dyes unequally, causing non-linear response in complex mixtures.

Which dye is routinely used for albumin assays and at what absorbance maximum?

Bromocresol purple (BCP); λmax ≈ 603 nm

In which patient group can BCP underestimate albumin and why?

Patients with renal insufficiency—altered albumin or a tightly bound substance hinders BCP binding.

Which reagent is used in the direct colorimetric assay for total globulins?

Glyoxylic acid in the presence of Cu²⁺ under acidic conditions.

At what UV wavelength is protein concentration estimated via intrinsic absorbance?

280 nm (aromatic amino acids Trp/Tyr absorb).

On what principal property does electrophoresis separate proteins?

Net electrical charge (and to some extent molecular size/shape).

If the buffer pH is higher than a protein’s pI, toward which electrode will it migrate?

Toward the anode (it carries a net negative charge).

Define a protein’s isoelectric point (pI).

The pH at which the protein’s net charge is zero and it does not migrate in an electric field.

Which two major proteins are absent from serum protein electrophoresis patterns and why?

Hemoglobin (inside RBCs) and fibrinogen (consumed during clotting).

What can a decreased A/G ratio indicate clinically?

Low albumin production/loss or increased globulins (e.g., multiple myeloma, chronic inflammation).

What principle underlies immunofixation electrophoresis (IFE)?

Antisera precipitate and ‘fix’ specific immunoglobulins in the gel after electrophoretic separation.

Which body fluids must be concentrated before IFE analysis?

Urine and cerebrospinal fluid (CSF).

State two advantages of capillary electrophoresis (CE) over traditional gel methods.

Rapid, high-resolution separations with automation and minimal sample volume requirements.

At which wavelength are proteins usually detected in CE and what absorbs there?

≈ 215 nm; peptide bonds absorb UV light.

What problem can surface charges in silica capillaries cause and how is it minimized?

Electroendosmosis; it is reduced by chemically modifying the capillary wall to neutralize charges.

What protein property allows isoelectric focusing (IEF) to achieve very high resolution?

Proteins focus at the exact pH equal to their pI within a stable pH gradient.

What is the typical pH range of commercial IEF gradients?

Approximately pH 3 to pH 10.

Proteins differing by how little in pI can be resolved by IEF?

Differences as small as 0.02 pH units.

How is the albumin-to-globulin (A/G) ratio calculated?

A/G ratio = Albumin / (Total protein − Albumin)