IC4 Flashcards
How is haemoglobin glycated?
Glycation of Hb takes place non-enzymatically: A condensation reaction btw glucose and amino end of the beta chain in Hb.
Which immunoassay method is used for DAC2000 HbA1c kit?
Immunoagglutination inhibition method; HbA1c (in patient’s blood) competes with agglutinator to bind to latex coated anti-HbA1c mouse monoclonal Ab.
What is the agglutinator used in DAC2000 HbA1c kit?
Synthetic polymer containing multiple copies of immunoreactive portion of HbA1c
Structure of insulin
110 amino acids polypeptide; Consists of an alpha & beta chain
Which cell produces pre-proinsulin?
beta cells in Islets of Langerhans of pancreas
How is pre-proinsulin converted to a mature insulin?
Removal of signal peptide from pre-proinsulin forms proinsulin. Formation of disulfide bonds btw A & B chain. Cleavage of proinsulin releases C peptide, forming a mature insulin.
Structure of MATURE insulin
chain A (21 amino acids) + chain B (30 amino acids) joined by 2 disulfide bonds
How is porcine and bovine insulin different from human insulin?
Porcine (pork) insulin: Single aa variation at B30;
Bovine (Beef) insulin: 3 aa variations at A8, A10 & B30
Disadvantages of using porcine/bovine insulin
- Slight differences in aa sequence could lead to immune response (lipoatrophy, local allergic reaction)
- Proinsulin: not totally removed during purification could cause immune response (lipoatrophy, local allergic reaction)
Production of recombinant human insulin
Proinsulin method:
1. Methionine codon (ATG start codon) chemically synthesized and attached to 5’ end of proinsulin cDNA. 2. Chemical cleavage by cyanogen bromide (CnBr) to remove Met residue from proinsulin polypeptide chain. 3. Proinsulin chain is subjected to folding process to allow intermolecular disulfide bond formation.
4. C-peptide cleaved by enzymes (trypsin and carboxypeptidase) to yield human insulin.
Properties of insulin
- Insulin has intrinsic ability to form dimers and higher order associated states (due to
favorable hydrophobic interactions) - In the presence of zinc, insulin is able to form zinc containing hexamer. Hexamer dissociates into monomers at site of injection when injected concentration falls to physiological levels.
Examples of rapid/ ultra acting insulin
Insulin Lispro/ Aspart/ Glulisine
Duration of action for rapid acting insulin
5 hours
Example of Intermediate acting Insulin Preparations
Neutral Protamine Hagedorn (NPH) insulin (AKA isophane insulin); available as Insulatard/ Humulin; cloudy suspension
What is NPH composed of?
Regular insulin and protamine (highly basic 30 amino acid peptide) co-crystallized with zinc in neutral pH (phosphate buffer) -> neutral crystalline insulin suspension.
