HPLC, high performance liquid chromatography, proteins, polynucleotides, peptides, reversed-phased chromatography, high, performance, liquid, chromatography, purification, RPC, synthetic, hormones, antigens, regnier, biologically active proteins, biology, chemistry, analytical, analytical chemistry, size-exclusion chromatography, SEC, ion-exchnage chromatography, IEC, polyacrylamide gel electrophoresis, PAGE, membrane, membrane protein, Oligonucleotides, Transfer Ribonucleic Acids, tRNA, Messenger RNAs, mRNA, Deoxyribonucleic Acids, DNAs
HPLC of Proteins and Polynucleotides
The following section discusses the use of HPLC in the separation and purification of proteins, peptides, and polynucleotides.Peptides: The use of reversed-phased chromatography (RPC) revolutionized peptide purification. Initially, synthetic peptides were purified, as were hormones and antigens. Use of RPC has become a common and important step in synthetic peptide production. More recently, RPC has been used to purify natural sequences. Although analytical columns are used to carry out the process, the procedure can be preparative in nature due to the limited amount of "active" proteins in tissue (Regnier, 1983). Some other advantages are that recovery of post-purification biological activity and reformation of secondary or tertiary structure after exposure to RPC are favored due to the abbreviated size of the peptides. Crude tissure extracts may be loaded directly onto the RPC system and mobilized by gradient elution. Rechromatography under the identical conditions is always an option if further purification is warranted or necessary. RPC can also be utilized in the process of protein structure determination. The normal procedure of this process is 1) fragmentation by proteolysis or chemical cleavage; 2) purification; and 3) sequencing. Recovery of hydrophobic fragments can be a problem (Regnier, 1983). A common mobile phase for RPC of peptides is a gradient of 0.1% trifluoroacetic acid (TFA) in water to 0.1% TFA in an organic solvent, such as acetonitrile, since the organic solvent 1) solubilizes the peptide, 2) allows detection at approximately 230-240 nm, and 3) can evaporate away from the sample (Regnier, 1983) .Biologically Active Proteins: The use of size-exclusion chromatography (SEC) and ion-exchange chromatography (IEC) is well-suited for use with biologically active proteins, such as enzymes, hormones, and antibodies, since each protein has its own unique structure and the techniques may be performed in physiological conditions. Full recovery of activity after exposure to the chromatography may be achieved, and currently, availability of SEC columns is diverse enough to allow fractionation from 10 to 1000 kilodaltons (Regnier, 1983). Extremely basic or hydrophobic proteins may not exhibit true SEC character since the columns tend to have slight hydrophobicity and anionic character. The use of gradient elution with the IEC column is favorable because of equivalent resolution as polyacrylamide gel electrophoresis (PAGE) and increased loading capability when compared to SEC.Less common is the use of liquid affinity chromatography (LAC) in applications of proteins. Interaction is based on binding of the protein due to mimicry of substrate, receptor, etc. The protein is eluted by introducing a competitive binding agent or altering the protein configuration which facilitates dissociation.Membrane Proteins: Membrane proteins are either peripheral (situated on the outer surface) or integral (partially span, entirely span, or lie completely within the membrane). The lipophilicity of the bilayer conveys the lipophilic character (i.e., hydrophobic amino acids) of the proteins within the membrane. RPC would be a logical choice in analysis and purification of these proteins, but IEC is also employed. One group has reported using formic acid and ethanol as mobile phase on a RPC system to ultimately (and successfully) chromatograph fragments of bacteriorhodopsin (Gerber, 1979).Another procedure used in the separation of membrane proteins is the use of nonionic detergents, such as Triton X-100, or protein solubilization by organic solvents with IEC.Oligonucleotides: Purifications can be achieved using RPC and IEC. For RPC, C18 columns can separate both derivatized and underivatized oligomers with up to 11 nucleotides and can be loaded with up to 1 mg sample (Haupt, 1983). Migration is dependent on chain length, phosphate groups, hydrophobic protecting groups, and chemistry of the purines and pyrimidines; thus, oligodeoxyadenylates migrate faster than oligodeoxythymidylates of equal length(Haupt, 1983).Separation by IEC can be accomplished using anion-exchange columns utilizing silica-based columns with quaternary amines. This method can separate mixtures with up to 20 bases. The columns tend to exude a hydrophilic nature, and use of organic solvents (methanol, acetonitrile) can aid this situation by decreasing any hydrophobic interactions and thereby relegate most interactions based on charge only. Therefore, order of migration will start with the shorter chains and longer chains follow.Transfer Ribonucleic Acids (tRNAs): Separations can be achieved for oligonucleotide chains of up to 50 bases on a system known as RPC-5, which contains trioctylmethylamine adsorbed onto a hydrophobic plastic particle (Wells, 1983).The column combines hydrophobicity and ion-exchange, but flow-rate must be decreased and analysis time is lengthened. Separation of tRNA can be accomplished by gradient elution where increasing organic fraction is added to the mobile phase. Separations can also be achieved by using descending organic solvent gradients in a RPC system, and by SEC for aminoacylated and nonaminoacylated tRNAs due to palpable differences in configuration (Wehr, 1979).Messenger RNAs (mRNAs): Reports which cite purification of mRNAs are scarce. In one study, various RNAs where eluted through a RPC column using acetonitrile as the mobile phase; amid the results, there were isolated mRNAs that produced a single peak on the chromatogram (Simonian, 1982). SEC can also be used in this separation.Deoxyribonucleic Acids (DNAs): Similar techniques used to separate large proteins can also be used in the fractionation of DNA (Kato, 1982) RPC-5 is a viable alternative, especially when isolating bacterial restriction fragments (Patient, 1979) . Percent recovery after elution is approximately 69-70, and fragments generally elute according to size (shorter chains before longer ones). Using gradient elution, the technique may separate fragments up to several thousand base pairs
.Regnier, F.E. Analytical Chemistry, 1983, Vol. 55, p. 1298.Regnier, p. 1299.Regnier, p. 1299.Regnier, p. 1299.Gerber, G.E.; Anderegg, R.J.; Herlihy, W.C.; Gray, C.P.; Beimann, K.; Khorana, H.G.; Proc. Nat'l Acad. Sci., 1979, Vol. 76, p. 227.Haupt, W.; Pingoud, A.; J. Chromatogr., 1983, Vol. 260, p. 419.Haupt, p. 419.Wells, R.D.; Hardies, S.C.; Horn, G.T.; Klein, B.; Larson, J.E.; Neuendorf, S.K.; Panayotatos, N.; Patient, R.K.; Selsing, E.;Methods Enzymol., 1980, Vol. 65, p. 327.Wehr, C.T.; Abbott, S.R.; J. Chromatogr., 1979, Vol. 185, p. 453.Simonian, M.H.; Capp, M.H.; Second Inter. Symp. HPLC of Prot. Pept. and Polynucl., Baltimore, MD, 1982, Paper #404.Kato, Y.; Nakamura, K.; Hashimoto, T.; Second Inter. Symp. on HPLC of Prot. Pept. and Polynucl., Baltimore, MD, 1982, Paper #403.Patient, R.K.; Hardies, S.C.; Larson, E.; Inman, R.B.; Maquat, L.E.; Wells, R.D.; J. Biol. Chem., 1979, Vol. 254, p. 5548.
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HPLC, high performance liquid chromatography, proteins, polynucleotides, peptides, reversed-phased chromatography, high, performance, liquid, chromatography, purification, RPC, synthetic, hormones, antigens, regnier, biologically active proteins, biology, chemistry, analytical, analytical chemistry, size-exclusion chromatography, SEC, ion-exchnage chromatography, IEC, polyacrylamide gel electrophoresis, PAGE, membrane, membrane protein, Oligonucleotides, Transfer Ribonucleic Acids, tRNA, Messenger RNAs, mRNA, Deoxyribonucleic Acids, DNAs