Purification of human α<sub>1</sub>‐antitrypsin by affinity chromatography on sepharose bound concanavalin A

Murthy, Rama J.; Hercz, Albert

doi:10.1016/0014-5793(73)80842-7

Cited by 52 publications

(17 citation statements)

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“…Partition chromatography has a high sample capacity and the resolving power of the column depends on its length, the number of plates, the relative amount of sample loaded, plus the selectivity of the different loading and eluting buffers used in the experiment (Ning et al, 2008;Tucholska et al, 2009). Many proteins were purified from blood fluids using sequential chromatography (Duckert, Koller, & Matter, 1953;Kanai, Raz, & Goodman, 1968;McConnell & Mason, 1970;Murthy & Hercz, 1973;Poffenbarger, 1975;Tollefsen, Majerus, & Blank, 1982). Proteins have been examined by partition chromatography prior to MS/MS using resins such as: Heparin (Tollefsen, Majerus, & Blank, 1982;Capila & Linhardt, 2002;Saito & Munakata, 2007;Lei et al, 2008); weak anion exchangers such as DEAE (Marshall et al, 2003; strong anion exchangers such as Quaternary Amine (Vasileva, Jakab, & Hasko, 1981;Sahab, Iczkowski, & Sang, 2007;Kovac et al, 2008); strong cation exchangers like propyl sulfate (Pieper et al, 2003a), carbohydrate binding columns like concanavalin A (Kass, Ratnoff, & Leon, 1969;Murthy & Hercz, 1973); size fractionation, hydrophobic interaction (Ruhaak et al, 2008), size exclusion, hydrophilic zwitterionic resins (Intoh et al, 2009), protein G resin (Neubert & James, 2009), size exclusion or gel filtration (Ratnoff, Kass, & Lang, 1969;Gao et al, 2005a;Hortin et al, 2006;Tucholska et al, 2007) or a battery of disposable preparative resins Tucholska et al, 2009).…”

Section: Partition Chromatography Of Proteins and Peptidesmentioning

confidence: 99%

Mass spectrometry of peptides and proteins from human blood

Zhu

Bowden

Zhang

et al. 2010

Mass Spectrometry Reviews

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It is difficult to convey the accelerating rate and growing importance of mass spectrometry applications to human blood proteins and peptides. Mass spectrometry can rapidly detect and identify the ionizable peptides from the proteins in a simple mixture and reveal many of their post-translational modifications. However, blood is a complex mixture that may contain many proteins first expressed in cells and tissues. The complete analysis of blood proteins is a daunting task that will rely on a wide range of disciplines from physics, chemistry, biochemistry, genetics, electromagnetic instrumentation, mathematics and computation. Therefore the comprehensive discovery and analysis of blood proteins will rank among the great technical challenges and require the cumulative sum of many of mankind's scientific achievements together. A variety of methods have been used to fractionate, analyze and identify proteins from blood, each yielding a small piece of the whole and throwing the great size of the task into sharp relief. The approaches attempted to date clearly indicate that enumerating the proteins and peptides of blood can be accomplished. There is no doubt that the mass spectrometry of blood will be crucial to the discovery and analysis of proteins, enzyme activities, and post-translational processes that underlay the mechanisms of disease. At present both discovery and quantification of proteins from blood are commonly reaching sensitivities of ∼1 ng/mL.

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Section: Partition Chromatography Of Proteins and Peptidesmentioning

confidence: 99%

Mass spectrometry of peptides and proteins from human blood

Zhu

Bowden

Zhang

et al. 2010

Mass Spectrometry Reviews

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“…One batch, corresponding to 25 U was dissolved in 1.0 ml distilled H 2 0 and used as indicated in legends to Fig. 1 and 2 For the purification of human a, -antitrypsin our earlier procedure [22] was used with certain modifications. As starting material only plasma aliquots, with a specific inhibitory potency of higher than 1.4 mg trypsin inhibited per ml, were used.…”

Section: Methodsmentioning

confidence: 99%

The Inhibition of Proteinases by Human α₁‐Antitrypsin

Hercz

1974

European Journal of Biochemistry

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The inhibition of human plasmin by a,-antitrypsin was time dependent and the rate of inhibition rapidly decreased with decreasing temperature.The effect of substrates on the inhibitions of trypsin and chymotrypsin was investigated. Theoretically the final degree of inhibition should be the same, whether the substrate is added to the proteinase-inhibitor system before or after the formation of the complex. The inhibition of chymotrypsin by a,-antitrypsin and of trypsin by commercial soybean trypsin inhibitor was in line with the prediction, with either casein or synthetic esters as substrates. However, in the trypsin-a,-antitrypsin system the degree of inhibition depended on the order in which the reactants were mixed. With both casein and p-tosyl-L-arginine methyl ester as substrates, inhibition was high when trypsin was preincubated with the inhibitor and the substrate was added later, and low or absent when the proteinase was added to the mixture of inhibitor and substrate.In the inhibition mixtures of plasmin, trypsin and chymotrypsin with a, -antitrypsin, distinct changes were detected by electrophoresis in polyacrylamide gel. The unreacted inhibitor diminished or disappeared and reaction products were formed at rates corresponding to rates of inhibition. a,-Antitrypsin, the most abundant proteinase inhibitor in human serum, exhibits a broad range of specificity toward proteinases. It is known to inhibit trypsin [I -41, chymotrypsin [2-41, elastase [3,5-81, plasmin [4,9-131, thrombin [4], collagenases [14,15] According to several authors the inhibition of trypsin and chymotrypsin by a,-antitrypsin is stoichiometric and is complete in a very short time [l -41. In contrast, the inhibition of kallikrein and plasmin is not strictly stoichiometric [4], is slow [3,4,10,11,16], and strongly temperature dependent [ 161. Acylation of a,-antitrypsin with maleic anhydride abolishes its trypsin-inhibiting capacity [ 19,201 suggesting that one or more lysine residues play a part in the binding process.Rimon et al. found that while inhibition of trypsin and chymotrypsin was not retarded in the presence of substrate, the inhbition of plasmin and thrombin was [4].I examined the reactions of plasmin, trypsin and chymotrypsin with a,-antitrypsin by inhibition studies. The effects of substrates on the inhibition of chymotrypsin were in good agreement with theoretical predictions, but on the inhibition of trypsin were not.The inhibition of the three different proteinases by a,-antitrypsin was accompanied by electrophoretically detectable changes, and the rate of these changes was about the same as the corresponding rate of inhibition.Some of these observations were included in a previous abstract [21].

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“…After equilibration, the inhibitor containing eluate was chromatographed on a Cellex D column [30] in 20 mM acetate buffer, pH 5.8, containing 30 mM NaCl and eluted by linearly increasing the concentration of NaCl to 150 mM in a total of 1.5 1 of buffer.…”

Section: Purification Of Variant Zmentioning

confidence: 99%

“…The last two steps of purification consisted of chromatography on DEAE-cellulose [31] (described in Results) and preparative gel electrophoresis [30]. The latter was carried out in a Buchler Polyprep model in a column of 7.5% polyacrylamide gel of 11 cm In addition to the above, another preparation of variant Z was obtained by a somewhat different procedure.…”

Section: Purification Of Variant Zmentioning

confidence: 99%

The Purification and Properties of Human α₁‐Antitrypsin (α₁‐Antiprotease), Variant Z

Hercz

Barton

1977

European Journal of Biochemistry

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After three stages of preliminary purification, variant Z was chromatographed on a DEAEcellulose column. Upon elution with a linearly increasing concentration of NaCI, variant Z was recovered in two separate peaks, the first of which contained 81 % and the second 19 % of the total.The preparation corresponding to the first peak was homogeneous by various criteria. The trypsin and chymotrypsin inhibiting capacities and the specific antigenic activity of the preparation were nearly the same as those of an authentic sample of variant M. Variant Z contained 8 or 9 more glycine residues than variant M, but no appreciable difference was found between their carbohydrate contents. By analytical isoelectrofocusing the isoinhibitors of purified variant Z overlapped with those in the plasma of the donor and were cathodal to, but partially overlapped with purified variant M. After desialysation, the overlap between the different variants became complete, but variant Z contained a larger proportion of cathodal and smaller proportion of anodal components than variant M. Both variants formed five distinct isoinhibitor-protease complexes after incubation with trypsin and chymotrypsin and the corresponding complexes in the different variants completely coincided.Over 20 different phenotypes of human al-antitrypsin can be distinguished by different electrophoretic and isoelectrofocusing methods (discussed in [I]). Some of the alleles of al-antitrypsin leading to a decreased plasma level of this protein, namely Piz (Pi standing for proteinase inhibitor) PiM Malton [lo] and PiM Duarte [ I l l attract special attention because of their medical as well was their theoretical implications.An outstanding discovery of the past few years in this field was the recognition of massive intrahepatic accumulation of al-antitrypsin in carriers of the Piz allele [12-151.The eel-antitrypsin in the serum of ZZ homozygotes is characterized by a slower (cathodal) than normal electrophoretic mobility . Several authors attempted to bring these observations to a common denominator by suggesting that both would arise decreased plasma level and decreased (cathodal) electrophoretic mobility of this protein [20,21]. This suggestion gained further credence by the observation that sialyltransferase activities in some scl-antitrypsindeficient members of a family were appreciably decreased [25].While this suggestion has had attractive features, on the whole it turned out to be untenable. As pointed out lately [26,27] the liver inclusion bodies in xlantitrypsin-deficient individuals are localized in the rough endoplasmic reticulum but have never been seen in the Golgi system of the hepatocyte, hence any possible alteration in the carbohydrate composition of this glycoprotein would be secondary to a primary defect of the peptide chain biosynthesis.In this work we purified variant Z from the plasma of a ZZ phenotype donor and compared it in various ways with purified variant M to gain insight into the nature of the defect leading to a1-antitrypsin deficiency. MA...

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Purification of human α₁‐antitrypsin by affinity chromatography on sepharose bound concanavalin A

Cited by 52 publications

References 16 publications

Mass spectrometry of peptides and proteins from human blood

Mass spectrometry of peptides and proteins from human blood

The Inhibition of Proteinases by Human α₁‐Antitrypsin

The Purification and Properties of Human α₁‐Antitrypsin (α₁‐Antiprotease), Variant Z

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Purification of human α1‐antitrypsin by affinity chromatography on sepharose bound concanavalin A

Cited by 52 publications

References 16 publications

Mass spectrometry of peptides and proteins from human blood

Mass spectrometry of peptides and proteins from human blood

The Inhibition of Proteinases by Human α1‐Antitrypsin

The Purification and Properties of Human α1‐Antitrypsin (α1‐Antiprotease), Variant Z

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Product

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Purification of human α₁‐antitrypsin by affinity chromatography on sepharose bound concanavalin A

The Inhibition of Proteinases by Human α₁‐Antitrypsin

The Purification and Properties of Human α₁‐Antitrypsin (α₁‐Antiprotease), Variant Z