Fast chromatofocusing of human serum proteins with special reference to α1-antitrypsin and Gc-globulin

Fägerstam, Lars G.; Lizana, J.; Axiö-Fredriksson, Ulla-Britt; Wahlström, Lennart

doi:10.1016/s0021-9673(01)90923-9

Cited by 22 publications

(3 citation statements)

References 10 publications

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“…Additional studies by Lester et al (1976Lester et al ( ,1978b using IEF and crossed immunoelectrophoresis demonstrated three AFP isomers having PIS ranging from 4.8 to 5.2. Although the previously reported PI values for AFP isomers are slightly different from those presented here, it is generally known that chromatofocusing may not always yield the same PI for a given protein as IEF (Fagerstam et al, 1983). Thus, the differences observed may be technique related.…”

Section: Discussioncontrasting

confidence: 90%

Characterization of human alpha fetoprotein charge microheterogeneity during fetal development

Keel¹,

Harms²,

Leal³

et al. 1990

Molecular Reproduction Devel

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Aliquotes of human amniotic fluid (AF), fetal serum (FS), and cord blood (CB) were obtained as by-products of routine clinical diagnostic procedures at term or in the second trimester of pregnancy. When samples of CB were applied to a pH 5.5-4 chromatofocusing gradient, three isoforms of AFP could be resolved; a pl 4.57 form (isoform IA, 52% AFP), a pl 4.27 form (isoform IB, 43% AFP), and one species that was bound to the column but could be eluted with 1.0 M NaCl (isoform II, pl less than 4.00, 5% AFP). Term AF displayed a profile similar to that observed in term CB. When samples of 15-20-week gestation AF were chromatofocused, the immunoreactive AFP recovered was distributed between isoform IA and IB (60%) and isoform II (40%). FS and AF obtained from same pregnancy (23-26 weeks) displayed an identical chromatofocusing profile. Aliquotes of AF subjected to conA revealed 83% reactive variants compared with greater than 95% reactive variants for CB. FS displayed a conA profile identical to CB. When individual CB charge isoforms were isolated and subjected to conA analysis, greater than 97% of the AFP bound to conA. In contrast, when AFP isoform IA and IB were isolated from midgestation AF, approximately 22% of the AFP did not bind to the lectin while 100% of isolated AFP isoform II eluted as the reactive variant. These data suggest that human AFP exists as at least three charge and two lectin variants and that the charge profile may change during fetal development.

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Section: Discussioncontrasting

confidence: 90%

Characterization of human alpha fetoprotein charge microheterogeneity during fetal development

Keel¹,

Harms²,

Leal³

et al. 1990

Molecular Reproduction Devel

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“…Further, as shown by Wakefield et al (1984), the elution range of a particular protein on chromatofocusing could vary depending on the sharpness of the gradient, presence of salt, or degree of column equilibration. The elution pH on chromatofocusing can be made to be near the pi if a suitable protocol is first established (Fagerstam et al, 1983). Obviously this is not a requisite for separating two particular proteins as shown by the good separation of esterase B from tonin obtained here.…”

Section: Discussionmentioning

confidence: 90%

Purification and characterization of a serine protease (esterase B) from rat submandibular glands

Khullar¹,

Scicli²,

Carretero³

et al. 1986

Biochemistry

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A new protease has been purified to homogeneity from rat submandibular gland homogenate by using DEAE-Sephadex chromatography, chromatofocusing, aprotinin-Sepharose affinity chromatography, and high-performance liquid chromatography. The enzyme has been named esterase B, since it represents the second major esterolytic peak on DEAE-Sephadex chromatography of submandibular gland homogenate. It is an acidic protein (pI = 4.45) with an apparent molecular weight of 27 000. It is heat-stable and has an optimum pH of 9.5. Esterase B hydrolyzed the synthetic substrates tosyl-L-arginine methyl ester and Val-Leu-Arg-p-nitroanilide (S2266). It also cleaved dog plasma kininogen to produce a kinin, identified as bradykinin on reverse-phase high-performance liquid chromatography. Esterase B, however, is only a weak kininogenase, since it had only 5% of the kininogenase activity of equimolar concentrations of glandular kallikrein and had no effect on rat mean blood pressure or on the isolated rat uterus. Esterase B activated plasminogen and had caseinolytic activity. It was inhibited by aprotinin, soybean trypsin inhibitor, lima bean trypsin inhibitor, phenylmethanesulfonyl fluoride, antipain, leupeptin, and p-tosyl-L-lysine chloromethyl ketone. On double immunodiffusion, when reacted with kallikrein and tonin antisera, esterase B showed partial identity with kallikrein but not with tonin. On immunoelectrophoresis against kallikrein antisera, esterase B formed a precipitin arc at a position different from that of kallikrein. Esterase B appears to be a trypsin-like serine protease having some homology with glandular kallikrein.

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“…Establishment of chromatofocusing as a high-performance separation technique was done by Hutchens et al 632 who used PEI-functionalized silica anion exchangers for analytical and preparative separation of estrogen receptor isoforms at good resolution and recovery. Coincidentally, Fägerstam et al 633 were working with polymer-based mixed strong/weak anion exchangers for fast highresolution chromatofocusing of human serum proteins, and for studying protein microheterogeneity. Chromatofocusing does not necessarily have to be carried out on anion exchangers.…”

Section: Chromatofocusingmentioning

confidence: 99%

High‐Performance Liquid Chromatography of Biological Macromolecules

Irgum¹

2000

Encyclopedia of Analytical Chemistry

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Fast chromatofocusing of human serum proteins with special reference to α1-antitrypsin and Gc-globulin

Cited by 22 publications

References 10 publications

Characterization of human alpha fetoprotein charge microheterogeneity during fetal development

Characterization of human alpha fetoprotein charge microheterogeneity during fetal development

Purification and characterization of a serine protease (esterase B) from rat submandibular glands

High‐Performance Liquid Chromatography of Biological Macromolecules

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