Chemical Attributes of Albumin that make it a Useful Serum-Free Media Supplement:
Albumin is a highly soluble, 69 kDa, acidic protein. It can bind anionic, cationic and neutral molecular species. Albumin has both high affinity, and secondary binding sites for many molecules. Ligands bound to their primary site are typically non-reactive. Ligands bound to secondary sites are typically reactive.
Amino Acid Sequences of Human and Bovine Albumin Precursors: The following are generally accepted sequences, however variants and mutations also exist. Bovine serum albumin preprotein (607) is two amino acids shorter than human serum albumin preprotein (609). The first 18 amino acids of albumin precursors provide secretion signals; the next 6 amino acids are propeptides, the albumin human and bovine albumin chains contains 585 (25-609) and 583 amino acids (25-607), respectively. The N-terminal tripeptides, DAH or DTH, form the Copper/Nickel binding pocket.
Human Albumin Amino Acid Sequence:
MKWVTFISLLFLFSSAY SRGVFRR DAH KSE VAHRFKDLGE ENFKALVLIA FAQYLQQCPF EDHVKLVNEV TEFAKTCVAD ESAENCDKSL HTLFGDKLCT VATLRETYGE MADCCAKQEP ERNECFLQHK DDNPNLPRLV RPEVDVMCTA FHDNEETFLK KYLYEIARRH PYFYAPELLF FAKRYKAAFT ECCQAADKAA CLLPKLDELR DEGKASSAKQ RLKCASLQKF GERAFKAWAV ARLSQRFPKA EFAEVSKLVT DLTKVHTECC HGDLLECADD RADLAKYICE NQDSISSKLK ECCEKPLLEK SHCIAEVEND EMPADLPSLA ADFVESKDVC KNYAEAKDVF LGMFLYEYAR RHPDYSVVLL LRLAKTYETT LEKCCAAADP HECYAKVFDE FKPLVEEPQN LIKQNCELFE QLGEYKFQNA LLVRYTKKVP QVSTPTLVEV SRNLGKVGSK CCKHPEAKRM PCAEDYLSVV LNQLCVLHEK TPVSDRVTKC CTESLVNRRP CFSALEVDET YVPKEFNAET FTFHADICTL SEKERQIKKQ TALVELVKHK PKATKEQLKA VMDDFAAFVE KCCKADDKET CFAEEGKKLV AASQAALGL
Bovine Albumin Amino Acid Sequence: (source page)
MKWVTFISLLLLFSSAYS RGVFRR DTH KSE IAHRFKDLGE EHFKGLVLIA FSQYLQQCPF DEHVKLVNEL TEFAKTCVAD ESHAGCEKSL HTLFGDELCK VASLRETYGD MADCCEKQEP ERNECFLSHK DDSPDLPKLK PDPNTLCDEF KADEKKFWGK YLYEIARRHP YFYAPELLYY ANKYNGVFQE CCQAEDKGAC LLPKIETMRE KVLASSARQR LRCASIQKFG ERALKAWSVA RLSQKFPKAE FVEVTKLVTD LTKVHKECCH GDLLECADDR ADLAKYICDN QDTISSKLKE CCDKPLLEKS HCIAEVEKDA IPENLPPLTA DFAEDKDVCK NYQEAKDAFL GSFLYEYSRR HPEYAVSVLL RLAKEYEATL EECCAKDDPH ACYSTVFDKL KHLVDEPQNL IKQNCDQFEK LGEYGFQNAL IVRYTRKVPQ VSTPTLVEVS RSLGKVGTRC CTKPESERMP CTEDYLSLIL NRLCVLHEKT PVSEKVTKCC TESLVNRRPC FSALTPDETY VPKAFDEKLF TFHADICTLP DTEKQIKKQT ALVELLKHKP KATEEQLKTV MENFVAFVDK CCAADDKEAC FAVEGPKLVV STQTALA
Albumin Complexes: At the functional level, albumin binds and delivers other molecules to cells in culture. The ability of albumin to support in vitro cell growth is largely determined by the type and quantity of nutrient ligands that it carries. A good appreciation of this can be gained by reviewing aspects of specific albumin ligand complexes.
Fatty Acid Binding: Fatty acids, such as linoleic, linolenic and oleic acid are insoluble in aqueous solutions and must be delivered to cells by a carrier molecule. Circulating albumin typically carries one or two free fatty acids. The binding of fatty acids also helps to stabilize the albumin. The activity of albumin as a cell culture supplement is partially dependent upon the specific fatty acids it binds and delivers to the cells.
Metal Binding: Zinc and copper are present in serum. They are important to the health of cells and are required components of cell culture. A large proportion of zinc in serum is bound to albumin. Copper atoms can undergo univalent redox reactions and catalyze the formation of free radicals. This feature makes copper toxic to cells. In vivo, the potential toxicity of extracellular copper is mitigated when it is bound to albumin. There is one high affinity site for copper per albumin molecule. When copper is bound to this site, it does not participate in the redox reactions associated with free radicals. Albumin binds other divalent cations, such as Ca, Mg, Mn, Cd, Co, and Ni.
Mixed Disulfides or Albumin: Human and bovine albumins contain an unpaired sulfhydryl at position 34 in their primary sequences. This sulfhydryl group often forms a covalent link with other sulfhydryl molecules such as cysteine or glutathione. Cysteine is not very stable in cell culture. It is easily oxidized to cystine and other oxidation products. By forming a protein mixed disulfide with cysteine and glutathione, HSA and BSA help to protect these molecules from oxidation and improve their availability for cells.
Pyridoxal Binding: Pyridoxal and its phosphate, pyridoxal-5’-phosphate, react non-enzymatically with amino acids to form Schiff bases. In aqueous solutions, especially in the presence of iron, these Schiff bases are unstable and result in the degradation of amino acids. Albumin binds pyridoxal at a site near its N-terminal. The binding of pyridoxal keeps it from reacting with and destroying amino acids in vitro.
Riboflavin and Tryptophan: Riboflavin can complex with tryptophan in aqueous solutions. In the presence of light this complex decomposes into toxic products. Riboflavin and its phosphate, flavin monophosphate, are bound and protected from degradation by albumin. Albumin has a single binding site for tryptophan.
Many other molecules bind to albumin under physiological conditions. These include, but are not limited to anions, drugs, and hormones.