albumin research in the 21st century
TRANSCRIPT
Biochimica et Biophysica Acta 1830 (2013) 5351–5353
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Preface
Albumin research in the 21st century
1. Early albumin research
It is important to recognize that most of our current knowledge ofserum albumin stems from many centuries of research. In the days be-fore serum albumin was recognized, scientists and physicians studied“albuminous material” in fluids of the body. For instance, Hippocratesof Cos, known as The Father of Modern Medicine, in his Aphorisms in400 B.C. noticed that the urine foamed in patientswith renal disease [1].
A millennium later, the Swiss physician, Paracelsus, who had a repu-tation for being arrogant but was a credible scientist, pioneered the useof chemicals in medicine, and reported turbidity in urine with additionof vinegar [2]. Frederick Dekkers, publishing in Leiden, The Netherlands,in 1694, compared the urine of consumptives to milk, being coagulablewith heat and acetic acid [3]. Nearly a century later, in 1764, DominicoCotugno of Bari treated the severe dropsy of a soldier with potassiumtartrate and provoked a massive diuresis, the urine of which uponheating became a white mass, “like egg albumin” [4].
In 1839, the eminent physiologist Henry Ancell at St. Georges Hospi-tal, London gave detailed lectures on the blood [5]. He accuratelyreported the content of albuminous substance or “albumen” (= totalprotein) in blood serum as 71 g/l. He refuted the proposal of M. Denisof Switzerland that albumen is “merely fibrine in solution”.
Suspicion was growing by 1886 that the “albumen” of serumcontainedmore than one protein component. G. Kauder pursued obser-vations that the addition of a salt, magnesium sulfate, to bovine bloodserum causes serum globulins to precipitate; these are retained on filterpaper, while the filtrate yields serum albumin upon dialysis [6]. Kauderemployed a saturated ammonium sulfate solution in place of magne-sium sulfate, and showed that incremental additions brought precipita-tion of additional globulin fractions and that “serumalbumin” remainedsoluble at 50% saturation.
2. Detection of serum albumin in blood
P.E. Howe, of the Rockefeller Institute in Princeton, NJ, in 1921 devel-oped salt fractionation into thefirst clinical assay for albumin [7]. Sodiumsulfate replaced ammonium sulfate since workers used the Kjeldahlnitrogen procedure tomeasure the protein. Howe studied the conditionscarefully, and found that the sodium salt gave better precipitation thanmagnesium sulfate, that a temperature of 37 °C is required and that21.5 g of anhydrous sodium sulfate per 100 ml of solution (1.5 M)gives an optimal separation of albumin and globulins.
Another assay involving precipitation took advantage of albumin'sunusual tolerance for acid conditions that would denature most pro-teins. The resilience to acidity is probably based on its 17 disulfidebonds aligned in a linear order. In 1932, J. Race published such a proce-dure to determine serum albumin with acid acetone [8]. In 1957, J.R.
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Debro et al. modified this approach to precipitate serum proteins with1% trichloroacetic acid, and then specifically dissolve the albumin with96% ethanol [9]. Addition of sodium acetate brings down the albumin,or the total globulins may be determined in the acid precipitate. Thisprocedure was adapted as a clinical method, although not a very“user-friendly” one, now superseded by dye-binding procedures.
Physical chemical methods contributed to the knowledge of serumalbumin. T. Svedberg of Uppsala in 1934 studied plasma proteins bythe new technique of ultracentrifugation and found albumin tomigratethe most slowly, at 4 S (“Svedberg units”), gamma globulins at 7 S andfibrinogen at 19 S [10]. This separation allowed isolation of albumin andshowed that it is a single protein of molecular weight near 70,000 Da,but is not practical as an assay. A. Tiselius in Svedberg's laboratorypioneered electrophoresis, the migration of charged molecules in anelectric field [11]. By this technique, he found serum proteins to sepa-rate into liquid boundaries of albumin, the fastest component, and α-,β-, and γ-globulins. Zone electrophoresis, on paper or polyacrylamideor agarose gel, now allows actual separation of protein bands. Theseare visualized by fixation and staining with colored dyes. On a prepara-tive scale, albumin can be obtained by elution from beds of starch. Theanalytical method is widely used in clinical laboratories for the detec-tion of abnormal bands such as myeloma proteins and for quantifyingof the electrophoretic components.
Serum albumin will bind many smaller molecules, especially thosethat are hydrophilic and negatively charged. This binding causes a“protein effect” first noticed with the pH indicator dye, methyl orange[12]. It is due to a structuralmodification of the dyemolecule that changesits optical spectrum and with excess dye can give a measure of the con-centration of the host albumin. The procedure is simple and now uses anumber of dyes. HABA (2-(4′-hydroxybenzeneazo)-benzoic acid), oneof the first, suffered from competitive binding by drugs and interferencebybilirubin [13].Methods usingbromocresol green [14], andbromocresolpurple [15], now dominate in clinical laboratories, and are sensitiveenough to measure the small amounts of albumin in cerebrospinal fluidand urine.
Albumin is a good antigen, and was used as early as 1938 as amodel to study the precipitin reaction [16]. Titers of 5 mg antibodyper ml can be prepared in rabbits; the molar ratio of BSA/antibodyis 4:1. Studies with monoclonal Fab fragments have identified atotal of 13 epitopes or antigenic sites in the HSA molecule [17]. Thestrong antigenicity allows the use of immobilized anti-albumin anti-bodies in affinity chromatography to isolate albumin and albuminfragments from tissue extracts [18]. The most sensitive assays for al-bumin now are immunoassays, which employ binding of antibodyto albumin detected by turbidity, nephelometry or displacement ofradioactive, fluorescent or enzyme-labeled albumin; concentrationsof albumin as low as 1 mg/L can be measured in urine.
5352 Preface
3. Characterization of albumin
Historically a major boost to our knowledge of albumin occurredwhen the USA entered WWII. The need for a stable substitute forwhole blood to treat shock on the battlefield caused the HarvardPhysical Laboratory in Boston to undertake preparation of serumalbumin for this purpose. The prominent protein physical chemist,E.J. Cohn, directed the Laboratory that he staffed with a team of expe-rienced albumin workers. They chose fractionation with ethanol,which they removed by lyophilization (freeze-drying) in contrast tosalts, and used low temperatures to minimize denaturation. TheirCohn Method 6 [19], which has been a mainstay of commercialfractionation, employed increasing alcohol concentrations at −5 °C.with varying pH and ionic strength to bring down 4 intermediate glob-ulin fractions. The next fraction “Fraction V”, precipitated at 40% ethanoland pH 4.8; the yield was 29.9 g albumin of 36.3 g in a liter of plasma.Fraction V was packaged as a 5%, 20% or 25% g/L solution and pasteur-ized for 10–11 h at 60 °C. The addition of 40 mM octanoate and40 mM acetyltryptophan provided protection against denaturation.
This Fraction V is the standard HSA used intravenously. By FDAspecification, it is 96% albumin and is stable for 5 years under refrig-eration. The same procedure can prepare bovine Fraction V, a cheaperprotein preparation, which is widely used in physical chemistry studiesand cell culture. A sad tale is that initially the Cohn laboratory preparedBSA rather thanHSA because of plentiful sourcematerial. Severe immu-nologic shock and even death occurred when this BSA was first givenintravenously, and the switch to HSA took place immediately, withthe assistance of the American Red Cross in providing plasma.
The use of Cohn fractionation to isolate large amounts of serumalbumin from blood plasma greatly aided its direct study. Earlystructural studies revealed that the protein to be composed of a sin-gle peptide chain, even following alkylation of all 17 of the disulfidebonds, and to possess an N-terminal Asp residue [20]. Specific cleav-ages allowed the isolation of large fragments. Meloun and co-workers in Prague obtained seven fragments following cleavage ofhuman albumin at its six methionines with cyanogen bromide.They determined the amino acid sequences of these with theEdman degradation in 1975 [21]. J.R. Brown's group in Texas alsofound the entire sequence of human albumin in the same year,using numerous tryptic peptides [22], and published the completesequence of bovine serum albumin (which contains only four me-thionines yielding five large fragments) as an abstract in 1975. Thebovine and human sequences are 76.8% identical. Comparison ofsequences of 17 serum albumins and related proteins appears inRef. [18] (pp144–145).
The sequence of the albumin gene, unlike that of the protein,appeared all at once rather than from fragments. R.M. Lawn et al. in1981 [23], and A. Dugaiczyk and coworkers in 1986 [24] publishedthe sequence of human albumin. Dugaiczyk's group reported 19,011nucleotides, including some 1817 nucleotides prior to the Cap site,14 introns and 15 exons. Exons 1 and 14 are partly translated. Exon15 is not translated at all but contains the signal for polyadenylationto form the polyA tail. Polymorphisms appear in the human codingsequence; Dugaiczyk's group [24] found nine substitutions of basepairs compared to the sequence published by Lawn et al. [23], buteight of them were silent when translated to amino acid residues.The article presented in this issue by Kragh-Hansen et al. presents afuller discussion of known albumin isoforms.
4. Current albumin research
The first X-ray crystal structure of human albuminwas published byX.M. He and D. Carter in 1992 [25]. Now over 50 human albumin struc-tures are now solved, providing snap-shots of serum albumin interactingwith a range of biologically important molecules that include fattyacids and drugs. In this regard, the work of S. Curry deserves special
mention [26]. In 2012, we saw the publication of the first non-humanalbumin structures, with the elucidation of bovine, equine andrabbit serum albumin structures [27,28]. Physical approaches suchas NMR and EPR spectroscopy are now shedding light on howalbumin “behaves” in solution. Serum albumin is often used (and hasbeen for many years) as a model in protein folding studies. Thefolding/unfolding of albumin, like other proteins, occurs in a stepwisefashion [29].
Arguably, albumin's most impressive feat is its ability to reversiblybind and transport a vast array of small molecules within the circula-tion. These include fatty acids, drugs and metal ions. The study ofsmall molecule binding is an extremely active area of albumin research.Articles detailing insights into the ligand-binding properties of serumalbumin that have been gathered using approaches including NMRspectroscopy, mass spectrometry, isothermal titration calorimetry, aswell as chromatographic and spectrophotometric methods featured inthis issue.
Modern proteomic methods, including advances in protein sepa-ration techniques and mass spectrometry have recently led to asurge in our knowledge of serum albumin metabolism and the chem-istry that occurs at its surface. The free thiol group of albumin (Cys34)undergoes a variety of redox reactions forming oxidized, nitrosylatedand guanylated derivatives, mixed disulfides and even albumin di-mers. In commercial HSA preparations, Cys34 is 15% bound to ahalf-cystine and 8% bound to nitrous oxide. About 8% of albumin mol-ecules have been non-enzymatically glycated at Lys residues, 4% aremissing the C-terminal leucine and 3% lack the N-terminal Asp-Ala[30]. The level of non-enzymatic glycation of albumin in vivo can bea useful clinical tool. The level of glycation rises in patients with dia-betes and, since albumin turns over faster than hemoglobin (27 versus120 days), its detection indicates the level of blood sugar in a shorterperiod than the glycation of hemoglobin. As mentioned above,albumin is measured in biological fluids using various methods.These include the use of albumin “dip-sticks” (which are dye-based),immunoassays and techniques such as HPLC and mass spectrometry(or combinations of the two), which can potentially distinguish be-tween derivatives/modified forms.
Human albumin is used clinically in the treatment of burns, shockand blood loss. It is also present in pharmaceutical preparations (suchas drug formulations and vaccines) and in cell culture media. Large-scale production of recombinant human albumin using yeast andplant-based expression systems is helping to meet the demand [31,32].Other biotechnological approaches include exploiting albumin's long cir-culatory half-life to enhance the pharmacokinetics and biodistribution ofdrugs, therapeutic proteins and nucleic acids. Thesemolecules are eitherbound to or fused with albumin. Similarly, albumin nanoparticles thatpossess the ability not only to transport small molecules but also toenable the controlled release of their bound cargo are currently beingdeveloped for in vivo use.
References
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Theodore Peters, Jr. is Research Scientist emeritus at TheMary Imogene Bassett Hospital (affiliated with Columbia Uni-versity) in Cooperstown, New York. He received a bachelor'sdegree summa cum laude in chemical engineering from LehighUniversity followed by graduatework at theMassachusetts In-stitute of Technology. Between service in the U.S. Navy as asubmarine communications and electronics officer in WWIIand in the KoreanWar, he obtained a Ph.D. in Biological Chem-istry from the Harvard Division of Medical Sciences. His thesisadvisor was Nobelist Christian B. Anfinsen. Upon studying thefate of 14C-amino acids in liver slices, he observed that a majorpart (25%) appeared in a secreted protein that proved to be se-rum albumin. With this finding, he became an albumin aficio-
nado and investigated its structure, function and metabolismfor most of his career. He taught at medical schools at the University of Pennsylvania andHarvard thenmoved to Cooperstown. Here he spent 33 years in basic research and 25 yearsin emeritus status. After retirement, he authored a book “All about Albumin. Biochemistry,Genetics andMedical Applications”, and has continued to publish papers on albumin genetics.In the laboratory, he held an NIH research grant for 28 years, longest in the blood studies di-vision. He is amember of the American Chemical Society, the American Society for Biochem-istry andMolecular Biology, the Protein Society, the American Society for Cell Biology, SigmaXi and Phi Beta Kappa, and is a Past President of the American Association for Clinical Chem-istry. Locally, he was named as “Environmentalist of the Year” in 2006 and for 40 years hasbeen Chairman of the CooperstownWastewater Treatment Board.
Alan J. Stewart is a Lecturer in Molecular Medicine at theUniversity of St Andrews in Scotland, UK. He graduatedfrom the University of Edinburgh with a BSc (Hons) de-gree in Biochemistry in 1999, and a Ph.D. in 2003. Dur-ing his doctoral studies he developed a strong interestin serum albumin where, under the supervision of PeterJ. Sadler, he examined the protein's metal-binding andredox properties. Following this, he took up a postdoc-toral position at the Roslin Institute to study skeletalbiochemistry, before moving to the MRC Human Repro-ductive Sciences Unit in Edinburgh as a Career Develop-ment Fellow. In 2009 he joined the School of Medicineat the University of St Andrews to establish his own re-
search group. His research is largely focussed on thevarious roles that metal ions play in the body. A key interest is their circulatorytransport (particularly by albumin) and their emergence as important mediatorsof disease.
Theodore Peters Jr.The Mary Imogene Bassett Hospital, Cooperstown, NY 13326, USA
E-mail address: [email protected].
Alan J. StewartSchool of Medicine, University of St Andrews, St Andrews KY16 9TF, UK
Corresponding author.E-mail address: [email protected].