Haematology - Haemoglobin

Question 1

Electrophoresis

Answer 1

Method of analysing molecules by measuring their migration in an electric field; support used as sieve through which molecules migrate; carried out on cellulose acetate gel strips held by bridge in trisglycerine buffer at 9.5 pH

Question 2

Where? What? Prevalence in Blood and Cells?

Answer 2

In RBCs (erythrocytes); carry O2 from lungs to tissues & transfer CO2 from tissues to lungs; 3.5 - 5x10^12 RBCs/L; each RBC = 640 mil Hb molecules

Question 3

Normal conc. in adults? Production and components?

Answer 3

120-165 g/L, 250 mg/kg produced and destroyed in body every day; each gram Hb = 3.4 mg Fe; haem synthesized in mitochondria, globin in rER

Question 4

Haem (Where? Structure? Behaviour?)

Answer 4

Also in myoglobin, cytochromes, peroxidases, catalases); photoporphyrin ring with central Fe atom (usually in ferrous (Fe2+) format); able to reversibly combine with O2 in pockets of globin protein

Question 5

Globins - Composition

Answer 5

Encoded by several genes in a couple of clusters (alpha and beta); protein product of genes combine with haem to form diff haemoglobin molecules; 8 functional globin chains produced; alpha and zeta encoded by alpha cluster (zeta, alpha1 and alpha2) & the beta, gamma, Delta, epsilon encoded by beta globin gene cluster (epsilon, Ggamma, Agamma, delta2, beta); proportions in healthy adult: HbA (alpha2beta2 tetramer) 96-98%, HbA2 (alpha2delta2) 1.5-3.2%, HbF (alpha2gamma2, 0.5-0.8%)

Question 6

HbA

Answer 6

secondary -> 75% alpha and beta helices in alpha form; quaternary -> approximate sphere, hydrophilic surface (charged polar side chains), hydrophobic core, haem groups within pockets

Question 7

Binding of O2 to Fe2+

Answer 7

Cooperative (change in confirmation of other subunits); positive bc affinity of remaining haem groups for O2 is increased (sigmoidal nature of O2 association)

Question 8

Changing pO2

Answer 8

Hemoglobin is oxygenated so pO2 ~ 100 torr (98% sat); passing through circulation where pO2 ~ 20 torr, sat 32%; 66% potential O2 binding sites contribute to O2 transport (hypothetical non-cooperative = 38%, positive cooperative enables Hb to deliver almost 2x O2)

Question 9

Myoglobin

Answer 9

One haem group/molecule; greater affinity for O2, saturating at lower pO2 values; no cooperativity so it is poor at releasing O2 at same conditions

Question 10

Spectrophotometry

Answer 10

Oxyhaemoglobin = 2 main peaks (540 and 580 nm); deoxy = single peak, 560 nm; used when checking respiratory status of newborn infants

Question 11

Pulse oximetry

Answer 11

Non-invasive way of measuring O2 sat; relies on difference in absorbance of oxy- and deoxyhaemoglobin; dipped onto thin appendage & emits light @ 660-940 nm (red visible & infrared); oxy -> absorbs more infrared but less red; transmitted light @ each wavelength is corrected for pulse of arterial blood by subtraction of minimum absorbance

Question 12

Carboxyhaemoglobin (CoHb)

Answer 12

Generated by the binding of carbon monoxide (200x affinity, 0.2% can lead to death)

Question 13

Methaemoglobin (MetHb)

Answer 13

Generated when Fe2+ oxidised to Fe3+; greatly impaired O2 binding = bluish/chocolate color if present @ high levels; low levels cause O2 dissociation curves to shift left = tissue anoxia (bc O2 not readily released by MetHb; methaemoglobin reductase makes it back into haemoglobin); methaemoglobinaemia can be hereditary (haemoglobin M resistant to reduction) or acquired following chemical exposure (ie aniline dyes/p-chloroaniline, nitrates, local anaesthetics (benzocaine))

Question 14

Haemoglobin Concentration with Absorbance

Answer 14

@ 540 nm; within reasonable range, Beer-Lambert law holds & absorbance is linearly proportional to concentration; graph -> y = A540, x = [Hb] mg/ml; HbA more negatively charged than HbS because of a point mutation changing glutamate (hydrophilic, negatively charged) to valine (hydrophobic, uncharged)