AA & Protein Analysis

proteins fxns

1. structural

2. transport

3. hormonal

4. catalytic

5. antibodies etc

conjugated proteins contain a ___ group ie

prosthetic

metalloprot, glycoprot, lipoprotein

protein size range

5,000 MW - 1 million

most are 12-36K MW (100-300 aa)

the ___ group determines most of the properties of the aa

R-

as pH inc → aa becomes more ___

neg charged

essential aa are ones that humans cannot synthesize ie

Leu

Ile

Val

Met

Phe

Trp

Thr

Lys

His(Arg)

all naturally occurring aa in prot are the _ configuration

L

measuring protein methods

1. chem (dye binding)

2. activity (enzyme rxn)

3. immunoassay

4. electrophoresis (ELP)

5. mass spectrometry

method dep on protein & its conc

total protein assay (Biuret, dye binding)

Cu2+ form violet complex w peptide bonds → intensity is proportional to # bonds

• can modify w +tartrate or +iodide

• **dye binding tends to underestimate low MW proteins!! → only detects 3 - 12 g/dL range

what is the method for measuring total urine protein?

pyrogallo red -molybdate

urine has low protein so need more sensitive

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Benzethonium chloride → turbidity

• turbidimetry: photodetector is parallel to incoming light

• nephlelometry: photodetector is perpendicular …

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dipstick uses tetrabromphenol blue (changes to blue if albumin)

• much more sensi ~15 mg/dL

proteinuria

types

• glomerular

• tubular

• overload - used for detecting lots of monoclonal light chains in multiple myeloma

_ assays are susceptible to hook effect

turbidimetry

prozone = xs antibody (protein~body)

postzone = xs antigen

→ solution

• wash xs analyte

• dilution

why would airfuge not work for a lipemic sample that has a CRP test on it?

C-reactive protein would be lost in the lipid layer → do 1:20 dilution

enzyme unit is

U = 1 umol/min (substrate converted)

electrophoresis is based on

molecules electrical charge & size

anode is + charged

cathode is - charged

the isoelectric point is the _

pH at which a molecule is neutral charged

pI = (pka1 + pka2)/2

1. Alb: pre- & albumin

2. a1: a1-antitrypsin, alphafetoprotein

3. a2: haptoglobin

4. B1B2: tranferrin, beta lipoprotein

5. gamma: fibrinogen, CRP

normal vs MM PEL

acute phase reactant proteins include

1. C-reactive protein (CRP)

2. serum amyloid protein (SAP)

3. fibrinogen

4. mannose binding lectin/protein (MBL)

5. complement

stim by IL-6

immunofixation is used for what

(similar to PEL) uses antisera for IgG, A, M, light chains kappa, lambda in sep tracks to visualize specific Ig

if have light chains & no G/A/M → check for IgD/E, may just have light chains only

immunotyping

serum + antisera against heavy chains and light chains

disappearance of the abnormality in antisera-treated pattern → type of protein indicated???

isoelectric focusing

migration of charged particles through pH gradient (via ampholytes in gel)

migration stops when pH=pI

detected by immunofixation (oligoclonal banding)