Human serum albumin is present in human blood plasma, produced in the liver. An anionic 66.5-kDa single-chain non-glycoprotein composed of 585 amino acids and rich in aspartic acid and lysine, it's the most abundant protein constituting about half of total serum protein. It's a highly water soluble molecule and very stable with 17 disulphide bridges (see schematic image left). HSA's abundance means it exerts a significant colloid osmotic (oncotic) pressure and clinically is infused to replace lost fluid and help restore blood volume in trauma, burns and surgery patients (acting as a plasma expander). World wide use (has been) very high; amounting to 100's of tonnes p/a with typically 12-17 gm patient infusions. When I started working with this in 1979, its amino acid sequence was available (an heroic task prior to cDNA sequencing!), but little about its 3D structure was known.
HSA is usually isolated from blood plasma with bound fatty acids and bilirubin (see later). Its drug binding capacity means it plays an important role in pharmacodynamics. It displays many activities including antioxidant activity and lipophilic hormone binding. Low albumin levels are associated with poor disease prognosis. Glycated serum albumin is also a marker of glucose intolerance. Diseases associated with HSA are shown at the foot of this article.
It was clear from earlier work on the analogous bovine serum albumin that the protein's single chain was folded into at least 3 domains. This is evident from the loop structure shown in the schematic image of the main chain. Using limited proteolysis I was able to seperate and purify individual HSA domains and investigate their binding properties. It was clear from circular dichroism (CD) measurements that the protein chain was largely alpha-helical and the isolated domains retained this secondary structure and bining properties. But it was also clear that pH changes caused significant relative movement between domains. Bound bilirubin changed its conformation when HSA solution was acidified and tightly bound fatty acids could be recovered by lowering the pH in the presence of activated charcoal. Experience with isolated domains representing two-thirds and one third of the molecule was to prove useful later in working with recombinant HSA at Delta Biotechnology Ltd.
I took purified product to FISONS instruments where the firm was developing electrospray mass spectrometry instrumentation. Despite the instrument used being an early prototype I was "blown away " when the measured mass of Delta rHSA came within 2 to 3 proton masses of that calculated from the amino acid sequence. Had even one of the 17 disulphide bridges not been formed, we would have been able to detect the difference! For me, as a protein chemist used to obtaining rough protein molecular weights by gel electrophoresis, this was like Saul's revelation on the road to Damascus and I later becane a consultant to FISONS Instruments as my first consultancy contract.
Recalling the first structural studies I carred out on HSA at the MRC labs at Mill Hill, I suggested that Delta express recombinant forms of 1- and 2- domain HSA. A number of recombinant proteins were designed and successfuly secreted: rHSA 1-585 (full length); 1-387; 1-407 and 1-194. The rationale behind expressing shorted versions of HSA was that (in principle) with 2/3 smaller molecular weight less protein would have to be infused for a given colloid osmotic pressure (oncotic) effect. Patents were applied for but in fact this idea was not pursued in practice
Recombinant HSA was taken through to production in a purpose build pilot plant in Nottingham and a full quality assurance programme initiated in preparation for Phase I clinical trials. After various sales and acquistion, Delta IP and production of rHSA was taken up by ALBUMEDIX (Nottingham). Phase I comparability of recombinant human albumin and human serum albumin has been published: Bosse D1, Praus M, Kiessling P, Nyman L, Andresen C, Waters J, Schindel F. J Clin Pharmacol. 2005 Jan;45(1):57-67.
Albumedix are now marketing rHSA and partnering other businesses to use rHSA for applications such as gene and cell therapies, vaccine stabilisation, protein and peptide formulations and medical device coating. For much background since Delta, interested readers should visit ALBUMEDIX website. Research around the albumin molecule and its biology has increased dramatically during the past decade. Several albumin binding proteins and receptors have been identified, and it has been demonstrated that these play an important role in the transport of albumin between different compartments, as well as its internalization, degradation, salvage and recycling. Most well understood is the interaction of albumin with the neonatal Fc receptor (FcRn) and the impact that this receptor has on the long serum half-life of albumin (approximately three weeks).
- Sepsis HSA may benefit specific groups of hypoalbuminemic critically ill patients.
- Renal disease: Proteolytic HSA fragments (like the ones I created?), are present in nephrotic and diabetic patients.
- Antioxidant activity Albumin binds copper ions with high affinity and scavenges free radicals, offering a thiol group for covalent conjugation.
- Diabetes Higher than normal glycated albumin levels are associated with development of insulin resistance in healthy people Thus glycated albumin levels can serve as diagnostic of prediabetes.
- Hypoalbuminemia Associated with poor postoperative prognosis in patients, but HSA infusion does not seem to alter clinical outcome.
- Inflammation: An inflammatory prognostic index is calculated as C-reactive protein × (neutrophil / lymphocyte ratio)/serum albumin.
- Cirrhosis Albumin is used clinically for hepatorenal syndrome and peritonitis.
COVID-19: Inflamation led to submicron extracellular vesicles (MPs) derived from platelets and endothelial cells. Pre-incubation of these COVID-19 derived MPs with the phosphatidyl capping Annexin A5 abolished cytotoxicity, adhesion protein induction and neutrophil and DNA traps called NETS. MPs appeared to be a key factor on COVID-19 pathology and point towards Annexin A5 as a possible therapeutic.HSA is vital in maintaining a healthy circulation and organs. Excess amounts or deficiency of HSA, both potentially lead to adverse effects. Hugely studied and subject to large number of reports in the literature, as the prinicpal protein component of blood plasma, certainly more remains to be discovered!
- Physical and Binding Properties of Large Fragments of Human Serum Albumin. Michael J Geisow & Gilbert Beaven (1976) Biochem. J. 163 477-484
- Production of Recombinant Human Serum Albumin from Saccharomyces cerevisiae. Alan V Quirk, Michael J Geisow, John R Woodrow, Steven J Burton, P Carolyn Wood, Andrew D Sutton, Richard A Johnson & Neil Dodsworth (1989) Biotechnology and Applied Biochemistry 11 273-287
- Characterisation and Quality Assurance of Recombinant Human Serum Albumin by Electrospray Mass Spectrometry. Michael J Geisow, Roy Harris, Neil Dodsworth, Brian Green and Therese Hutton (1991) Techniques in Protein Chemistry II 54 567-572
- Mass Spectrometry in the Standardisation of Recombinant Products. M J Geisow (1994) in Genetic Stability and Recombinant Product Consistency Brown, F and Lubiniecki (eds) 83 129-133
- New Developments in Biochemical Mass Spectrometry. Michael Geisow (1996) Biologicals 21 125-129
- Electrospray mass spectrometry of proteins. Michael Geisow (1994) The Biochemist Briefing Papers 13 No. 3
- Patents applied for: 1. Colloid Osmotic Pressure regulating protein. 2. Wound-healing polypeptide. 3. Albumin fusion proteins.