Immunochromatography on insolubilized antibodies of very low affinity: application to immunoadsorbence of bovine alpha-fetoprotein.

Cross-reactive antibodies were utilized to prepare immunoadsorbents possessing a very low affinity to bovine alpha-fetoprotein (AFP). A goat anti-human AFP serum cross-reactive with bovine AFP was first depleted of antibodies reactive with bovine AFP in immunodiffusion. The remaining antibodies from this serum and gamma-globulin from a sheep antiserum against rabbit AFP, without prior absorption, were coupled to Sepharose. Chromatography of fetal calf serum on these adsorbents resulted in retardation of bovine AFP relative to other proteins. A major part of the AFP eluted from the columns with phosphate-buffered saline. The rest eluted as a sharp peak with a small quantity of 4 or 6 M urea. The elution of AFP with the initial column buffer has made it possible to prepare pure AFP that has not been subjected to the chaotropic elution buffers usually employed in affinity chromatography. Elimination of the washing step and the ease of elution has allowed purification of gram amounts of AFP. The fact that immunoadsorbents prepared from antibodies with no detectable reactivity in immunodiffusion still caused delayed elution in chromatography suggests that this procedure may be useful in search of proteins cross-reactive with a known protein.     

Affinity chromatography of human alpha-fetoprotein on immobilized estrogens

Human alpha-fetoprotein was isolated from abortive material with the help of affinity chromatography on immobilized estrogens. After butanol extraction from the abortive material human AFP obtained the ability for affinity binding with immobilized estrogens. The addition of estrogens to the extract of isolated AFP preparation and incubation with them did not lower AFP binding with immobilized estrogens during the experiments, using affinity chromatography. A 10% buffered aqueous butanol solution was most optimal for elution. The data obtained can suggest that AFP in biological fluids is bound to estrogens, and butanol extraction deestrogenizes human AFP. The mechanism of AFP binding to estrogens in vivo is, evidently, carried out with the help of specific unknown carrier, as AFP does not bind free estrogens.     

Rat alpha-fetoprotein: isolation, radioimmunoassay and fetal-maternal distribution during pregnancy.

A method is described for the isolation of mg quantities of two forms of rat alpha-fetoprotein (AFP) from amniotic fluid by preparative disc-gel column electrophoresis using a continuous elution system. AFP isolated by this method is suitable for use as an antigen, can be labelled for use in a radioimmunoassay and serves as a reference standard. The characteristics of a new antiserum to AFP are also described. The protocol for a radioimmunoassay is outlined which permits the measurement of AFP in several fetal-maternal physiological compartments throughout gestation. Levels of AFP in fetal liver and fetal plasma suggest that secretion of AFP from liver occurs soon after synthesis with minimal hepatic storage. The pattern for AFP in maternal serum parallels that observed in amniotic fluid and fluctuations in maternal serum levels of AFP appear to be buffered by AFP accumulation in amniotic fluid. Fetal clearance of AFP under normal conditions may be relatively constant from Days 11-20 of gestation since an amniotic fluid: maternal serum AFP ratio of 30:1 is maintained during this period.     

Chromatofocusing profile of purified human alpha-fetoprotein and albumin differs from those of crude samples: effect of protein concentration of the elution of the sample.

Chromatofocusing was utilized to characterize charge microheterogeneity of purified human alpha-fetoprotein (AFP) and human serum albumin (HSA). Crude cord blood samples yielded three isoforms: AFP-IA, IB, and II, with pIs 4.57 (52%), 4.27 (43%), and less than 4.00 (5%), respectively. In contrast, 10 micrograms of purified AFP or 250,000 cpm of 125I-AFP eluted entirely as isoform AFP-II. 125I-AFP focused in the presence of crude cord blood, amniotic fluid, adult male serum, or 25 mg purified HSA resulted in elution profiles similar to those of crude cord blood. Pure AFP focused along with 0.1, 1.0, 5.0, or 10 mg HSA showed a gradual shift from AFP-II to AFP-I. With greater than or equal to 5 mg HSA, isoform I was further resolve into AFP-IA and IB. Similarly, 250,000 cpm of 125I-HSA, which also eluted entirely as isoform II, showed a gradual shift to isoform I when increasing concentrations of unlabeled HSA were added. The resolution of isoform HSA-I in HSA-IA, IB, and IC was again improved with greater than or equal to 5 mg unlabeled HSA. When carrier proteins of varying pI values were chromatofocused along with purified AFP, it was observed that only those proteins with pIs in the range of AFP caused significant alteration in the relative distribution of AFP. We conclude that sample protein concentration and composition must be carefully considered when chromatofocusing is being used for purified samples and when the elution profiles of samples from different origins and varying protein concentrations are being compared.     

Characterization of human alpha fetoprotein charge microheterogeneity during fetal development.

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.     

Immunological demonstration of alpha-fetoprotein in uterine cytosol from immature rats

An immunological demonstration of alpha-fetoprotein (AFP) in uterine cytosol from immature rats is reported. The identity between serum AFP and the estrogen binding 4.5 S macromolecular complex of uterine cytosol was demonstrated by the use of a specific immunoadsorbent to AFP. Analysis of the sedimentation profile in glycerol gradients of uterine cytosol incubated with tritiated estrone or estradiol suggests that the total estrogen binding capacity of the 4.5 S complex is provided by AFP. Changes of AFP content in rat uterus with the age of the animals suggest that this protein is probably present in the cytosol as a serum contaminant.     

Determination of multiple redox-active compounds by high-performance liquid chromatography with coulometric multi-electrode array system

Studies of the antioxidant defense system and the related metabolic pathways are often complicated by cumbersome analytical methods, which require separate and multi-step extraction and chemical reaction procedures. Further, assaying multiple parameters is limited because of the usual small sample amounts. High-performance liquid chromatography coupled with a coulometric multi-electrode array system provides us high specificity and sensitivity to measure electrochemically oxidizable compounds in biological samples. In contrast to previously reported methods with two columns in series and a complex gradient elution profile, we have developed an automated procedure to simultaneously measure multiple redox-active low-molecular weight compounds that utilizes a single column with a simplified binary gradient profile. No other chemical reactions are necessary. In order to reduce the running time and yet achieve a reproducible retention time by the auto sampler injection, our gradient elution profile was modified to produce a shorter equilibration time, stable retention time, and a reduced cost per test.     

A structural basis for four distinct elution profiles on concanavalin A--Sepharose affinity chromatography of glycopeptides.

Twelve 14C-acetylated glycopeptides have been subjected to affinity chromatography on concanvalin A (Con A)--Sepharose at pH 7.5. The elution profiles could be classified into four distinct patterns. The first pattern showed no retardation of glycopeptide on the column and was elicited with a glycopeptide having three peripheral oligosaccharide chains: (abstract:see text). Such glycopeptides have only a single mannose residue capable of interacting with Con A--Sepharose; an interacting mannose residue is either an alpha-linked nonreducing terminal residue or an alpha-linked 2-O-substituted residue. The second type of profile showed a retarded elution of glycopeptide with buffer lacking methyl alpha-D-glucopyranoside (indicative of weak interaction with the column) and was given by glycopeptides with the structures: (abstract: see text) where R1 is either H or a sialyl residue. The third profile type showed tight binding of glycopeptide to Con A--Sepharose and elution as a sharp peak with 0.1 M methyl alpha-D-glucopyranoside; glycopeptides giving this pattern had the structures: (abstract: see text) where R2 is either H, glcNAc, Gal-beta 1,4-GlcNAc, or sialyl-Gal-beta 1,4-GlcNAc. These glycopeptides all have two interacting mannose residues, the mimimum required for binding to the column; one of these mannose residues must, however, be a terminal residue to obtain tight binding and sharp elution. The fourth profile type showed tight binding of glycopeptide to the column but elution with 0.1 M methyl alpha-D-glucopyranoside resulted in a broad peak indicating very tight binding; glycopeptides showing this behaviour had the structures: (abstract: see text) where R3 is either GlcNAc,Gal-beta 1,4-GlcNAc, or sialyl-Gal-beta 1,4-GlcNAc.Therefore it can be concluded that although a minimum of two interacting mannose residues is required for binding to Con A--Sepharose, the residues linked to these mannoses can either strengthen or weaken binding to the column.     

A 55-kilodalton accessory factor facilitates vitamin D receptor DNA binding.

The interaction of the vitamin D receptor with a vitamin D-responsive element (VDRE) derived from the human osteocalcin promoter in vitro has been shown to require a nuclear accessory factor (NAF) derived from monkey kidney cells. In this report we show that this factor is widely distributed in cells and tissues, including those that do not express the vitamin D receptor (VDR). NAF is required for VDR binding to a variety of known VDREs. VDR and NAF independently bind the VDRE weakly, as assessed by elution profiles generated during VDRE affinity chromatography. Together, however, both proteins coelute from this column with a profile that indicates a tighter strength of interaction. Analogous chromatography of the VDR derived from ROS 17/2.8 cells treated with 1,25-dihydroxyvitamin D3 in culture also reveals a dual profile of weak and strong binding, suggesting that in vivo modifications are unlikely to alter receptor DNA binding. NAF is a protein of 55 kDa, as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and cross-linking experiments suggest that the VDR and NAF together form a heterodimer on a single VDRE with a mol wt of 103 kDa. These data demonstrate that NAF is required for VDR binding to specific DNA in vitro and suggest the possibility that NAF may be required for the transactivation capability of the VDR in vivo.     

A single step procedure for purification of estradiol 17 beta-dehydrogenase from human placenta

A new and simple procedure for purification to homogeneity of the soluble estradiol 17 beta-dehydrogenase [E C.1.1.1.62] from human placenta has been developed. The purification is achieved in a single step by affinity-absorption of Cibachrome Blue F3G-A coupled to Sepharose and selective elution with NADP+. Homogeneous estradiol 17 beta-dehydrogenase has a specific activity of 7.2 units/mg. and has been purified 2400-fold with a 93% recovery.     

Comparative biochemistry of acid proteinase from animal origins

Acid proteinases from 17 tissues of 12 animal species were compared with respect to molecular weight, inhibition by pepstatin and activation by tripolyphosphate. Gel filtration of acid proteinases from protochordates and vertebrates showed a common elution profile and three peaks with mol. wts of -20,000, -45,000 and above 150,000 were detected with acid-denatured hemoglobin as substrate at pH 3.6. The main component of vertebrate acid proteinases was identified as cathepsin D. In the invertebrate acid proteinases, the elution profiles through gel filtration were characteristic to the tissues examined, and were not so distinct as those of vertebrates. Through a biochemical survey, the animal acid proteinase was discussed from a comparative point of view.     

Synthesis of alpha-fetoprotein by the pre-implantation and post-implantation bovine embryo

The synthesis of alpha 1-fetoprotein (AFP) was measured by radioimmunoassay in tissues and fluids of 19 bovine embryos (14-46 days of gestation) and in tissue cultures of 4 pre-implantation embryos (17-27 days) by incorporation of radioactive methionine. AFP was first detected in Day-14 trophoblasts and secretion of AFP into allantoic fluid occurred by Day 16. Embryonic tissues and fluids in pre-implantation and post-implantation embryos contained levels of AFP that were 550 to 1 500 000 times higher than those found in maternal serum (3.9-298 000 compared with 0.07-0.25 ng/mg protein). High levels of AFP were also found in uterine fluid which suggested significant transfer of this protein from the early post-implantation conceptus. The major sites of AFP synthesis were yolk sac and fetal liver. It is concluded that the synthesis of bovine AFP is not initiated by events associated with implantation.     

Isolation, Characterization, and Synthesis of AlphaFetoprotein from Neonatal Rat Brain

Monospecific anti-rat serum alpha-fetoprotein (AFP) IgG was coupled to cyanogen bromide-activated Sepharose-4B (4.5 mg/ml packed volume of gel) to yield an immunoaffinity matrix. The immunoaffinity column was used to isolate AFP from feto-neonatal rat brain. The purified AFP was immunologically and electrophoretically similar to serum AFP. It yielded a single band with a molecular weight of 70,000 on sodium dodecyl sulphate polyacrylamide gel electrophoresis. Polyacrylamide gel electrophoresis of the protein under nondenaturing conditions yielded two charge variants of AFP, reminiscent of AFP from feto-neonatal rat serum. The AFP was observed to bind estradiol with Ka = 5.8 X 10(8) M -1 and 1.3 X 10(8) M -1 by dextran-coated charcoal adsorption and Sephadex gel filtration techniques, respectively. Newborn rat brain cells linearly incorporated [14C]leucine into immunoprecipitable AFP during 6 h in culture. It is, therefore, concluded that feto-neonatal rat brain contains AFP similar to that present in fetal serum and that it may arise in brain as a result of its in situ synthesis.     

人胎儿肝内甲胎蛋白的成份及其对T.B淋巴细胞发育的影响

本文用免疫组织化学方法,分别在冰冻和石蜡切片上,对24例不同胎龄的胎儿肝比较研究了肝内 AFP~+细胞数量及其与 T,B 淋巴细胞之间的关系。结果发现 AFP 仅分布于肝细胞内,其他细胞阴性。不同胎龄的肝脏,AFP 的染色强度和阳性率不同。17周前的胎肝,AFP~+细胞最多以后逐渐减少。出生前的肝脏内只有少数 AFP~+细胞。AFP~+细胞的多少与 B 细胞分化发育无多大关系,但与 T 细胞似乎关系密切,两者呈负相关,即 AFP~+细胞多时,T 细胞很少,AFP~+减少时,T 细胞增加,提示 AFP 对 T 细胞具有抑制作用。同时也证明 B 细胞在胎肝内受 T 细胞的影响不大,主要依赖于肝脏的微环境。另外对 AFP 的生物学意义也进行了讨论。     

Selectivity of IMAC columns in trypsin inhibitor purification

The properties of an adsorbent and the parameters in an adsorption process affect the resolution of chromatographic purifications. This is reflected in the elution profile, which shows the relative affinity of different proteins for a specific adsorbent. In the work presented here, elution profiles for trypsin inhibitor were used to study the effects of the concentration of trypsin inhibitor, ionic strength of the protein solution, slope of the elution gradient, and the regeneration treatment of the chromatography column on the selectivity of the adsorbent Cellufine Chelate-Cu(II)(ida). Cytochrome c was used as a reference protein. Variations in the concentrations of trypsin inhibitor and in the ionic strength of the buffered solution did not have any effects on the elution profile. On the other hand, changes in the slope of the pH gradient used for elution caused shifting of the elution peaks toward lower values of the elution volume, resulting in the best strategy to modify the elution profile of the system. Finally, using a constant slope pH gradient of elution, the variation of the selectivity of the adsorbent for trypsin inhibitor when subjected to cleaning treatments with 0.5 N NaOH was studied. Appropriate cleaning practices used in industry were followed. The adsorbent showed only a slight tendency for resolution loss in the order of 2 x 10(-4) days(-1). The results presented here show a good stability of the adsorbent when compared to other biospecific adsorbents commonly used.     

Evaluation of the electroosmotic medium pump system for preparative disk gel electrophoresis

This paper describes an improved electroosmotic elution system for preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) based on the epochal idea of H. V. Tan et al. (Nucleic Acids Res. 1988, 16, 1921-1930). In this elution system, a semipermeable membrane, mounted under the gel terminal end, works as the elution pump as well as the partition of the elution chamber. We refer to this system as the "electroosmotic medium pump system." Operation of the constructed apparatus (3.6 cm i.d. disk gel column) and resolution of the protein bands were examined by separation of the model protein mixture (bovine serum albumin (BSA), ovalbumin, bovine carbonic anhydrase, soybean trypsin inhibitor) and purification of the membrane protein, dipeptidyl peptidase IV (DPP IV). The Spectra/Por 7 dialysis membrane provided a better flow profile for the elution buffer. The four model proteins of the protein mixture were able to be completely separated from each other and recovered without dilution. The maximum protein concentration of eluate achieved was 93 mg/ml, when applying a single component, BSA fraction V, as a sample. Furthermore, the multifunctional ectoenzyme, DPP IV, was purified in a single step.     

Hyperactive antifreeze protein from fish contains multiple ice-binding sites

Antifreeze proteins (AFPs) are produced to prevent freezing in many fish species that are exposed to icy seawater. There are a number of nonhomologous types of AFPs, diverse in both sequence and structure, which share the function of binding to ice and inhibiting its growth. We recently discovered a hyperactive AFP in the winter flounder and related species that is many-fold more active than other fish AFPs. Like the 3-4-kDa type I AFPs, it is alanine-rich and highly helical, but this 17-kDa protein is considerably larger and forms a dimer. We have sequenced the cDNA encoding this new AFP to gain insight into its structure and evolutionary relationship to the type I AFP family. The gene is clearly homologous to the righteye flounder type I AFP genes. Thus we have designated this protein "hyperactive type I AFP" (hyp-type I). The sequence of hyp-type I AFP supports a structural model in which two extended 195-amino acid alpha-helices form an amphipathic homodimer with a series of linked Ala- and Thr-rich patches on the surface of the dimer, each of which resembles ice-binding sites of type I AFPs. The superior activity of hyp-type I AFP may derive from the large combined surface area of the ice-binding sites, recognition of multiple planes of ice, and protection of the basal plane from ice growth.     

Selection of monoclonal antibodies for immunoaffinity chromatography: model studies with antibodies against soy bean trypsin inhibitor

To facilitate selection of monoclonal antibodies for immunoaffinity chromatography, an ELISA screening procedure was developed. The assay is based on the avidin-biotin system and provides a profile of the monoclonal antibody which is based on the binding characteristics of the antigen binding site when exposed to different elution reagents. The elution profiles of 5 monoclonal antibodies to soy bean trypsin inhibitor (SBTI) were determined and for 2 of the antibodies the results obtained in the ELISA were verified using column experiments. The affinity constants were determined for the same 5 monoclonal antibodies and no correlation was seen with the ease of elution. The elution profiles presented here are easily obtained and the results indicate that a general screening procedure for suitable combinations of antibodies and elution conditions can be carried out using an elution ELISA assay when modified as described herein.