Isoforms isoelectric

Heterogeneity. Natural aequorin is not a homogeneous protein it is a mixture of many isoforms having isoelectric points ranging from 4.2 to 4.9 (Blinks and Harrer, 1975). The isoform composition may vary to some extent by the purification method employed, due to uneven loss of isoforms during purification. Consequently, the properties of each preparation of aequorin may also vary. By anion-exchange... 

All cell-wall extracts contained several PME isoforms differing in their isoelectric points. The isoenzyme patterns changed significantly during cell ageing. 

In E. Coli bacterial lysates, the proteome (i.e., the full array of proteins produced) was analyzed by isoelectric focusing and mass spectrometry.97 A comparison of capillary electrophoretic separation and slab gel separation of a recombinant monoclonal antibody demonstrated that the precision, robustness, speed, and ease-of-use of CE were superior.98 Seventy-five proteins from the yeast ribosome were analyzed and identified by capillary electrophoresis coupled with MS/MS tandem mass spectrometry.99 Heavy-chain C-terminal variants of the anti-tumor necrosis factor antibody DE7 have been separated on capillary isoelectric focusing.100 Isoforms differing by about 0.1 pi units represented antibodies with 0,1 or 2 C-terminal lysines. 

Jochheim C., Novick S., Balland A., Mahan-Boyce J., Wang W.C., Goetze A., and Gombotz W.R. (2001), Separation of Enbrel (rhu TNFR Fc) isoforms by capillary isoelectric focusing, Chromatographia Suppl. 53, S59-S65. 

Somatropin is a protein having the structure (191 amino acid residues) of the major component of growth hormone produced by the human pituitary. Somatropin is produced by a method based on recombinant DNA technology. Recently the results of a collaborative study between 14 laboratories have been reported, in which a new CE method was compared with the existing test for isoform distribution by isoelectric focusing. The CE method was found to be superior to the method of isoelectric focusing. As a result CE is applied for identification... 

As with SDS-PAGE gel methods, gel-based isoelectric focusing (lEF) methods have been used for decades to determine isoelectric point pi), which is an intrinsic property of protein molecules. Some complex proteins have multiple charge isoforms with multiple isoelectric points. These isoforms are separated as multiple bands in the lEF gel method. However, like other gel method, the lEF gel has limitations it is not automated, not reproducible, and not quantitative for pi determination. It is also labor intensive and requires large volumes of toxic reagents for staining. 

E Contiero, R Ferrari, GM Vaselli, M Folin. Apolipoprotein A1 isoforms in serum determined by isoelectric focusing and immunoblotting. Electrophoresis 18 122-126, 1997. 

Isoelectric focusing of different samples of rhEPO (recombinant human erythropoietin).The different profiles of isoforms are observed in the different preparations. Lane 3 international standard. 

It had been known for some years that there is more than one form of apoA-I in plasma (El, L23, 07), when Nestruck et al. (N5) reported that four forms of apoA-I could be isolated by preparative flat bed isoelectric focusing. The two major forms in human plasma (referred to as apoA-Ij and apoA-I2 by Nestruck et al., but as isoforms or isoproteins 4 and 5 in this review (following references S9, Zl, and Z6), focus at pi 5.62 and 5.53, respectively, and contain 71 and 19%, respectively, of total apoA-I. All forms had an identical apparent Mr and common antigenicity to antisera against apoA-I. The amino acid analyses of isoforms 4, 5, and 6 resembled previously published apoA-I analyses (Bl, B43) and these isoforms activated purified lecithimcholesterol acyltransferase. 

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. 

The isoforms of apoE were first clearly demonstrated by Utermann et al., who showed on one-dimensional isoelectric focusing of VLDL apolipoproteins that there were four major isoforms of apoE (U3— U5). These were named, from acidic to basic, apoE-1, apoE-2, apoE-3, and apoE-4. Patients with Type I1 hyperlipoproteinemia (dysbetalipoproteinemia) were, in virtually all cases, deficient in apoE-3 (U3-U6). Only 27% of subjects had apoeE-4, but this band was not associated with any particular abnormality. However, individuals deficient in apoE-3 were also deficient in apoE-4 (U6). Utermann suggested that the genetic pattern of apoE isoforms could be explained if there was a single genetic locus for apoE which could produce three apoE phenotypes, i.e., apoE-N (normal), apoE-D (deficient, associated particularly with the Type III disorder), and apoE-ND (U4, U6). 

Fig. (11). Isoelectric focusing pattern for anti-lactose antibodies composed of 13, 16 and 21 Isoforms.

Table 1. Isoelectric Points for Anti-Lactose Isoforms...

The native molecular mass of aphid myrosinase, estimated from gel filtration, is approximately 97 kDa, while the molecular mass of the denatured and reduced protein is 53 kDa, estimated from SDS PAGE, and the subunit by MALDI-TOF mass spectrometry 54 kDa 500 Da. Thus, aphid myrosinase appears to be a dimeric protein, with identical subunits. The isoelectric points (pi) of aphid myrosinase are 4.90 and 4.95, the latter isoform being the most abundant. 

Usually, immunoglobulins are N-glycosylated at the Fc part in some cases, terminal neuraminic acid may be missing. This leads to slightly different isoelectric points of the various members in the family called isoforms. Therefore, an IgG preparation which exhibits a single band in sodium dodecyl sulfate (SDS) electrophoresis exhibits a characteristic band pattern in isoelectric focusing (Fig. 4). 

For the adsorption of antibodies, virtually all ion-exchangers compatible with proteins can be used. Anion-exchangers have been used extensively for the isolation of polyclonal IgG from human plasma and from that of different mammals in a single step with a high degree of purity in a single pass, as shown in Fig. 8. They have also been used to isolate isoforms of monoclonal IgG after optimization of adsorption and elution conditions to fit with isoelectric point properties of the antibody.91... 

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