Chromatography isoforms

Masuda, H., et al. (2003). Chromatography of isoforms of recombinant apoaequorin and method for the preparation of aequorin. Protein Expression and Purification 31 181-187. 

Hydrophobic interaction chromatography (HIC) can be considered to be a variant of reversed phase chromatography, in which the polarity of the mobile phase is modulated by adjusting the concentration of a salt such as ammonium sulfate. The analyte, which is initially adsorbed to a hydrophobic phase, desorbs as the ionic strength is decreased. One application demonstrating extraordinary selectivity was the separation of isoforms of a monoclonal antibody differing only in the inclusion of a particular aspartic acid residue in the normal, cyclic, or iso forms.27 The uses and limitations of hydrophobic interaction chromatography in process-scale purifications are discussed in Chapter 3. 

Stewart PM, Murray BA, Mason JI (1994) Human kidney 1 l/ -hydroxysteroid dehydrogenase is a high affinity nicotinamide adenine dinucleotide-dependent enzyme and differs from the cloned type I isoform. J Clin Endocrinol Metab 79 480-484 Taylor RL, Machacek D, Singh RJ (2002) Validation of a high-throughput liquid chromatography-tandem mass spectrometry method for urinary cortisol and cortisone. Clin Chem 48 1511-1519... 

Analysis of NRX isolated from brain by a-LTX-affinity chromatography (Petrenko et al. 1990 Petrenko et al. 1993) shows that out of the six main isoforms only NRX la binds toxin with sufficient affinity (Davletov et al. 1995), and this interaction strictly requires Ca2+ (Petrenko et al. 1990 Davletov et al. 1995). 

ApoD is found in association with LCAT and with apoA-I in the HDL fraction. Albers et al. used a specific antibody to apoD to remove all apoD by immunoadsorption chromatography from plasma about 64% of LCAT activity and 11% of apoA-I were also removed from plasma (A14). Purified apoD has an apparent Mr of 32,500, and appears as three isoforms on isoelectric focusing (pi 5.20, 5.08, and 5.00) (A14). An HDL apolipoprotein, Mr 35,000, has been thought to be apoD, and to be a cholesteryl ester transfer protein (i.e., to transfer newly synthesized esterified cholesterol from HDL to LDL) (C8). Cholesteryl ester transfer activity in plasma was removed by polyclonal immunoglobulin to apoD (C8, F10). However, Morton and Zilversmit (M41) were able to separate apoD and lipid transfer protein (i.e., the cholesteryl ester transfer protein, or lipid transfer protein I) by chromatography, and they showed that the removal of apoD from plasma by precipitation with specific antisera did not remove any lipid transfer activity. Albers et al. (A14) also showed that immunoadsorption with antibody specific for apoD removed all the apoD from plasma without removing any cholesteryl ester transfer activity. 

All the anti-carbohydrate antibodies purified by affinity chromatography are isomers of a different number of isoforms. The forms can be separated by electrofocusing and all have been found to have combining activity for the immunodeterminant of the same antigen by the method of coupled analysis of electrofocusing and agar diffusion [88], The isoforms of antimonosaccharide antibodies are shown in Fig. (48). 

Several isoforms of tyrosine protein kinase play roles in cellular growth and differentiation. The assay of Boutin et al. (1992), exploits the use of hydrophilic interaction chromatography to separate hydrophilic peptides. 

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