N-[(carbamoylmethyl)amino]ethanesulfonic acid improves phenotyping of alpha 1-antitrypsin by isoelectric focusing on agarose gel

The hereditary deficiency variants of alpha 1-antitrypsin that are associated with diseases such as emphysema are usually identified by use of isoelectric focusing on polyacrylamide gels. Agarose is a simpler, faster, safer, and more reliable medium for this, but resolution often is not as good. I describe a method in which the ultrathin agarose gel contains N-[(carbamoylmethyl)amino]ethanesulfonic acid as a "separator," to flatten the pH gradient and improve separation of the alpha 1-antitrypsin isoforms. The resolution obtained equals or surpasses that of conventional methods based on use of either polyacrylamide or agarose. Haptoglobin, which interferes with isoelectric focusing on polyacrylamide, does not interfere with this method; other advantages are also discussed.     

Isoelectric focusing in agarose: classification of genetic variants of alpha 1-antitrypsin

Polyacrylamide has been the matrix of choice for isoelectric focusing owing to the virtual absence of electroendosmosis in this medium. Certain inherent limitations associated with polyacrylamide have prompted some investigators to use low-electroendosmosis agarose for isoelectric focusing, but with limited success thus far. We have developed a method for isoelectric focusing in agarose for the classification of alpha 1-antitrypsin variants. Sera are applied directly to agarose gels containing a pH 4-5 ampholyte mixture, focused for less than 1 h, and directly immunofixed. Resolution of major bands is equivalent to polyacrylamide, and Pi M subtypes can be distinguished without the use of a separator. This application demonstrates the high resolution of isoelectric focusing in agarose, a more practical and convenient matrix than polyacrylamide.     

Genetic typing of alpha1-antitrypsin by immunofixation electrophoresis, identification of subtypes of Pi M.

Prolonged electrophoresis in alkaline agarose gels, followed by immunofixation, is a valuable addition to acid starch gel electrophoresis and isoelectric focusing for the genetic phenotyping of alpha1-antitrypsin. This technique is helpful in clarifying certain variants and in ascertaining types in serum and amniotic fluid samples with secondary changes. In addition, heterogeneity may be detected within the variants found at pH 4.95, analogous to hemoglobin polymorphism. Two "new" variants, PiMLamb and PiMBaldwin, have been detected by a combination of immunofixation electrophoresis and acid starch gel electrophoresis.     

The microheterogeneities of thyroxine-binding globulin and alpha 1 protease inhibitor (alpha 1-antitrypsin) are interrelated

Addition of 125I-thyroxine to serum allows autoradiography for thyroxine-binding globulin microheterogeneity to be carried out after isoelectric focusing has been performed to display (by protein stain) the heterogeneous bands of the alpha 1-antitrypsin (PI) system. Comparison of the protein stain for PI with the autoradiograph for thyroxine-binding globulin indicates that these two systems are interrelated with the major bands of the PI system corresponding to the bands on the autoradiograph. This correspondence holds for PI variants other than the common M type and in particular it holds for the deficient Z type in which the autoradiograph for thyroxine-binding globulin is strikingly different from normal. We conclude that the major cause of microheterogeneity of TBG is due to an association with the PI system under the conditions of isoelectric focusing as normally performed. Precipitation experiments with antisera to PI and TBG suggest that the complex between these biologically important globulins may occur under conditions other than isoelectric focusing, but further work will be needed to examine this possibility.     

Improved method for identifying alpha 1-antitrypsin Pi M subtypes by isoelectric focusing in agarose

We describe an improved method for the classification of alpha 1-antitrypsin variants by isoelectric focusing in agarose. Identification of the three Pi M subtypes can now be made by using a narrow-range carrier ampholyte (pH 4.2-4.9) and pretreating serum with dithioerythritol-iodoacetic acid to enhance band resolution. Phenotype results for two groups of Pi M homo- and heterozygotes are compared to illustrate the improved accuracy of the new method.     

Genetic variants of alpha 1-antitrypsin and hemoglobin typed by isoelectric focusing in preselected narrow pH gradients with PhastSystem.

In this method for automatically running and staining isoelectric focusing (IEF) gels, pre-made dehydrated polyacrylamide gels were rehydrated before assays run with the PhastSystem (Pharmacia LKB Biotechnology). The typing of genetic variants of hemoglobin and alpha 1-antitrypsin in narrow pH gradients (pH 6.7-7.7 and 4.2-4.9, respectively) was simple, convenient, and reproducible. The clinically important variants of alpha 1-antitrypsin (ZZ and SZ) were identified from serum or dried blood on filter paper. The fast screening of abnormal hemoglobin samples (HbS) for cases in clinical medicine was easily performed. The total analysis time for the phenotyping with conventional protein staining was approximately 60 min.     

Purification and characterization of immunosuppressive (IS) substance obtained from ascitic fluids of patients with gastrointestinal cancer

An immunosuppressive substance was isolated from ascitic fluids of patients with advanced colon cancer by means of ammonium sulphate precipitation and preparative isoelectric focusing. This was a glycoprotein with an isoelectric point of pH 2.7-3.3, a molecular weight of about 52,000, and a sedimentation coefficient of 4.0S. The substance showed a single band on ordinary disc electrophoresis, but it was separated into several bands by gel isoelectric focusing, due to the structural variety of the sugar moiety. The results of physicochemical analysis indicate that the amino acid composition of this glycoprotein was indistinguishable from that of alpha 1-acid glycoprotein (alpha 1-AG), but its molecular weight, its carbohydrate content, and composition were distinctly different. Furthermore, this glycoprotein was found to have higher immunosuppressive activity than that of alpha 1-AG in both the in vitro and in vivo assays. This glycoprotein, which we called "IS substance," is a cancer-related substance synthesized in cancer patients.     

Multiple isoelectric forms of bacterial penicillin-binding proteins: artefacts or genuine cellular components?

Some bacterial penicillin-binding proteins (PBPs) are reported to exist as multiple isoelectric forms. Other PBPs were examined by two-dimensional electrophoresis to establish whether multiple isoelectric forms are widespread amongst PBPs. Bacillus subtilis PBPs 3 and 5 and Escherichia coli PBPs 1b alpha and 1b gamma focussed as discrete spots with no evidence of multiple isoforms. However, E. coli PBPs 1b beta and 4 displayed isoelectric variants. The results are discussed in relation to current models of bacterial peptidoglycan structure.     

Determination of alpha1-antitrypsin phenotypes by isoelectric focusing in polyacrylamide gels.

A method for alpha1-antitrypsin phenotyping is described that uses isoelectric focusing in a pH gradient 3.5 to 5 with polyacrylamide as supporting medium. The method is fast, reproducible, and reliable and offers some advantages over the currently used starch-gel electrophoresis.     

Molecular characterization of the P and I variants of alpha 1-antitrypsin.

Two rare alpha 1-antitrypsin variants, Pi I and Plowell, originally defined at the protein level through isoelectric focusing, were characterized at the DNA level by the polymerase chain reaction and direct sequencing. The I variant was confirmed in one individual and three independent families to result from a CGC(Arg) to TGC(Cys) transition at codon 39, within exon II. In our population, the Pi I variant might be more common than expected. The Plowell allele was shown in one M3P heterozygous individual to be due to a GAT(Asp) to GTT(Val) change at codon 256, in agreement with a previous study based on hybridization with allele-specific oligonucleotides.     

Optimizing reference values for the measurement of alpha 1-antitrypsin in serum: comparison of three methods

We studied three methods (rate nephelometry, radial immunodiffusion, and trypsin-inhibitory capacity) for their ability to detect those individuals with a deficiency of alpha1-antitrypsin. The phenotype represented in 170 serum samples was determined by isoelectric focusing as the reference method. All three methods correctly identified Pi Z, Pi S, and Pi SZ phenotypes but varied in their ability to detect Pi MZ and Pi MS phenotypes. The rate-nephelometric method was the least sensitive in detecting Pi MZ and Pi MS variants because of the inappropriately low reference interval suggested by the manufacturer. We found that the three screening methods are comparable when the limiting values are properly selected. We suggest that the reference value for the rate-nephelometric method be increased from 0.85 g/L to 1.40 g/L to improve the sensitivity of the test.     

A null deficiency allele of alpha 1-antitrypsin, QOludwigshafen, with altered tertiary structure.

The most common deficiency allele of the plasma protease inhibitor alpha 1-antitrypsin (alpha 1AT) is PI*Z. Some rare deficiency alleles of alpha 1AT produce low but detectable amounts of plasma alpha 1AT (1-20% of normal), which can be differentiated by isoelectric focusing. Others, designated null (QO) alleles, produce no alpha 1AT detectable by routine quantitative methods. We have previously described a method using DNA polymorphisms, haplotypes, and polyacrylamide isoelectric focusing gels, to differentiate various deficiency alleles. Based on haplotypes, we previously identified, in eight patients, five different null alleles, four of which had been previously sequenced. We have now analyzed all 12 null alleles in these eight patients, using allele-specific oligonucleotide probes, and have identified six different null alleles. We have cloned and sequenced one of these, PI*QOludwigshafen, which has a base substitution in exon II, replacing isoleucine 92 in the normal sequence with an asparagine. This substitution of a polar for a nonpolar amino acid occurs in one of the alpha-helices and is predicted to disrupt the tertiary structure. A total of 13 different alpha 1AT deficiency alleles, 6 of them null alleles, have been sequenced to date.     

Clearance of alpha 1-antitrypsin isoelectric focusing patterns in blood samples treated with heparin.

Plasma samples, whose typing for alpha 1-AT is made difficult or impossible because of an excess of heparin used as anticoagulant, can be treated very effectively with protamine sulphate. The addition of this heparin antagonist results in complete clearance of the isoelectric focusing pattern.     

Altered isoelectric focusing of alpha 2-macroglobulin from plasma of patients with diabetes mellitus

Plasma from both type I and type II patients with diabetes mellitus displayed elevated alpha 2-macroglobulin trypsin-binding activity relative to normals. Column isoelectric focusing indicated that the average isoelectric point (pI) of the major form of crude alpha 2-macroglobulin was 6.6, 5.9, and 6.2 for normal, type I and type II subjects, respectively. Focused crude diabetic alpha 2-macroglobulin displayed significantly less recovered trypsin-binding activity relative to controls, particularly above a pI value of 6.0. Following preincubation with trypsin, the crude diabetic alpha 2-macroglobulin displayed a single isoelectric form (pI value of 5.3-5.5) and a substantial increase in recovered activity which were both comparable to normal alpha 2-macroglobulin. Purified alpha 2-macroglobulin from both normal and diabetic subjects displayed similar focusing profiles having a distribution of three forms with a pI value of 5.3-5.4 for the principal form. Trypsin-preincubated samples of purified alpha 2-macroglobulin displayed a single form with a pI value of 5.5-6.0. Preincubation of purified normal or diabetic alpha 2-macroglobulin with a plasma preparation devoid of alpha 2-macroglobulin from normal or diabetic plasma resulted in significantly lowered recovery of activity. All of the above studies suggest that the altered focusing properties observed for crude diabetic alpha 2-macroglobulin are due to components of the plasma rather than to alpha 2-macroglobulin itself.     

Cystic fibrosis alpha 2-macroglobulin protease interaction in vitro

alpha 2-Macroglobulin was purified from plasma of five cystic fibrosis patients and five normal controls. SDS gel electrophoresis of native alpha 2-macroglobulin from cystic fibrosis patients and normal donors showed identical subunit molecular weights, as did trypsin cleavage products. Cystic fibrosis and control alpha 2-macroglobulins were indistinguishable by isoelectric focusing and exhibited appropriate shifts in isoelectric point following binding of trypsin. The trypsin-binding capacities of control and cystic fibrosis alpha 2-macroglobulins did not differ, nor did the esterolytic activity of the trypsin-alpha 2-macroglobulin complexes.     

The isoelectric focusing of creatine kinase variants: I. The heterogeneity of creatine kinase in human heart cytosol and mitochondria

Creatine kinase isoenzymes in cytosolic and mitochondrial fractions from human cardiac tissues were studied by analytical and preparative isoelectric focusing (IEF), electrophoresis and immunoinhibition. Analytical IEF on agarose gels revealed many creatine kinase variants in human cardiac cytosol prepared by extraction with a hypotonic medium. The bands located at approximately pH 5.5 were shown to contain creatine kinase-MB and minute creatine kinase-BB bands by electrophoresis. Two bands which focused closely together in IEF (pH 6.85-7.0) showed an electrophoretic migration pattern similar to creatine kinase-MM. One of them (IP 6.85) showed a complete inhibition by anti-creatine kinase-M antibodies, whereas the other showed only 50% inhibition. Increasing the salt concentration of tris-HCl (0.1 mol/l) in the extraction medium resulted in additional creatine kinase variants. They were characterized by high alkaline isoelectric points and were not inhibited by anti-creatine kinase-M antibodies. These variants corresponded to two cathodic bands in electrophoresis. The treatment of washed mitochondria with phosphate buffer resulted in a release of mitochondrial variants with different isoelectric points, as shown by analytical IEF in agarose gels. The same pattern was obtained by using preparative IEF. Variants with high alkaline isoelectric points gave rise to two cathodic bands upon electrophoresis. These two bands resembled those present in cytosol after extraction with high salt concentration. No complete inhibition with anti-creatine kinase-M was observed in any of the eluates. The mitochondrial variants exhibited different affinities towards creatine phosphate and ADP. Variants with higher alkaline isoelectric points showed lower Km-values for these substrates than those with less alkaline isoelectric points.     

Alpha 1-antitrypsin phenotypes of healthy persons and chronic bronchitis patients

The paper is concerned with phenotyping of alpha 1-antitrypsin using a method of isoelectric focusing in the polyacrylamide gel with ampholine (pH-4-6) in 1000 healthy persons and 583 patients with chronic bronchitis. Phenotype M was registered in 96 and 95.71% of the cases, respectively. The occurrence of heterozygous phenotypes was practically similar among the healthy persons as well as among the patients (4.0 and 4.29%, respectively). It was concluded that the heterozygous carrying of an alpha 1-antitrypsin deficit gene was of no particular importance in the development of chronic bronchitis.     

alpha-1-antitrypsin (Pi) phenotypes in a Finnish population.

alpha-1-antitrypsin (Pi) phenotypes of 548 normal Finnish blood donors were determined by isoelectric focusing in polyacrylamide gel. The frequencies obtained (M, 93.3%; FM, 0.4%, GM, 0.2%; MS, 3.5%; MZ, 2.7%) differed from those reported earlier for a more restricted Finnish population but agreed better with the frequencies reported for other Scandinavian populations. The disagreement with the earlier report was attributed to differences in sampling and laboratory techniques. The present study confirms an earlier finding of a sarcity of fast Pi variants in the Finnish population. One M variant was found. The isoelectric focusing technique used in this study has several advantages over the methods previously described for Pi typing.     

Thin-layer isoelectric focusing of hemoglobin variants: Screening and determination of isoelectric points

The high resolving power of thin-layer isoelectric focusing was applied for screening some hemoglobin variants classified on the basis of their electrophoretic mobility in: electrophoretically slow variants (as Hb A₂), electrophoretically slow variants (as Hb S), electrophoretically fast variants (Hbs type J).An analysis of the variant compounds has been performed, and the corresponding pI values were determined in whole hemolysate.     

Capillary electrophoresis of hemoglobin.

Capillary electrophoresis (CE) has been used in a variety of in-house capillary isoelectric focusing (CIEF) and capillary zone electrophoresis (CZE) assays for the detection of hemoglobin (Hb) variants and the quantitation of HbA2 and HbF. A commercial kit has also been produced for the analysis of hemoglobin variants and thalassemia screening. Though CE methods have been shown to be able to detect many variants, final identification of the variant needs specialized testing such as DNA technology. Over the past 2 years, many instruments that had been used for these hemoglobin variant screening and thalassemia assays have been withdrawn from sale. Although CE HbA1c analysis is available, it cannot compete in turnaround time or cost with automated HPLC commercial instruments that give accurate HbA1c results in 3 or 4 minutes. Hence we do not anticipate a bright future for the analysis of hemoglobin by CE.