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1.
A new approach for separation, capillary affinity chromatography, is introduced for studying the interaction of heparin with antithrombin III and secretory leukocyte proteinase inhibitor. Heparin is covalently immobilized on the surface of an etched capillary through a silane spacer. The proteins are injected into the heparinized capillary, bound to the heparin, washed with buffer, eluted with sodium chloride in the same buffer using a pressure injection mode and eluting protein detected by absorbance. The resulting affinity separation is similar to that obtained from traditional affinity chromatography. The quantity of loaded protein in capillary affinity chromatography is at the nanogram level, offering an improvement over the milligram levels required for standard affinity chromatographic methods.  相似文献   

2.
The insulin-like growth factor (IGF)-binding proteins (IGFBPs) carry IGFs in serum and regulate their activity and bioavailability. The main IGFBP in serum, IGFBP-3, is known to form a 150-kDa complex with IGFs and the acid-labile subunit (ALS). We investigated the binding of IGFBP-3 to additional association proteins in human serum (IGFBP-3 APs). Ligand blots, column chromatography, and affinity cross-linking experiments revealed the specific binding of IGFBP-3 to at least three novel serum proteins. These techniques demonstrated the presence of proteins with molecular masses of 70, 100, and 150 kDa that bind IGFBP-3 with high affinity. Serum ALS migrated separately (at 88 kDa) from the novel IGFBP-3 APs (as evident by Western immunoblot), and bound IGFBP-3 weakly (by reverse ligand blots). We also demonstrated that large amounts of one of the IGFBP-3 APs and small amounts of ALS were coimmunoprecipitated with IGFBP-3 from human serum. Similar to ALS, these IGFBP-3 APs are acid labile and lose their IGFBP-3 binding capacity after exposure to low pH. We conclude that there are several serum proteins in addition to ALS and IGFs that bind IGFBP-3 with high affinity. These IGFBP-3 APs may serve as an additional reservoir of IGFBP-3 or modulate its functions.  相似文献   

3.
Radiofrequency (RF) catheter ablation is the curative treatment of choice for many cardiac arrhythmias. After RF ablation there is always a small localized endomyocardial necrosis, necessary to abolish the arrhythmia. We designed this study to determine the serum concentrations of several cardiac markers in patients who underwent RF catheter ablation. The study shows a higher frequency of increase of serum cardiac troponin I (cTnI) than of creatine kinase (CK), the CK MB isoenzyme (CK-MB), or myoglobin. A pathological value of cTnI was found in 47 of 51 patients (92%) in the ablation group. The area under the ROC curve for cTnI was 0.9375, significantly higher than for the other biochemical markers (0.86, 0.76, and 0.75 for CK-MB, myoglobin, and CK, respectively), with P <0.05. We conclude that the serum concentration of cTnI is the best biochemical marker for detecting the minor myocardial damage produced by RF ablation.  相似文献   

4.
We used three different electrophoretic techniques for the analysis of human plasma proteins: (i) two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), with sodium dodecyl sulfate (SDS) used only in slab gel electrophoresis; (ii) capillary isoelectric focusing (CIEF) with no denaturants; (iii) linear polyacrylamide (LPA)-filled capillary electrophoresis with SDS (SDS-CE). With technique (i), data on isoelectric point and molecular size of plasma proteins can be obtained. Techniques (ii) and (iii) are suited to obtain quantitative information on proteins. The separation principle used in technique (ii) is closely related to that used in the first dimension of technique (i), and that used in technique (iii) related to that in the second dimension of technique (i). Therefore, we could successfully correlate protein separation patterns obtained by 2-D PAGE and those obtained by capillary electrophoresis. The advantages of correlating data obtained by various electrophoretic techniques in the course of constructing a comprehensive database on human plasma proteins are discussed.  相似文献   

5.
Reversed-charge capillary zone electrophoresis (RC-CZE) has been developed as a clipping (proteolysis) assay for homodimeric protein recombinant human platelet-derived growth factor (rhPDGF-BB), a major serum mitogenic factor involved in subcutaneous wound healing. When expressed in yeast, the protein is excreted as a fully folded homodimeric protein consisting of two antiparallel B chains held together by two interchain disulfide bonds. During fermentation, internal proteolysis (clipping between residues Arg32 and Thr33) and C-terminal truncation (Arg32 and Thr109) may occur. Internal proteolysis yields three potential forms of rhPDGF-BB: intact (both B chains are intact), single-clipped (one B chain is clipped), and double-clipped (both B chains are clipped). Clipping also creates new C-terminal sites for further C-terminal truncations and leads to a very complex mixture of isoforms. Routine baseline resolution of these three forms by various modes of HPLC proved unsuccessful. When the disulfide bonds of antiparallel chains are reduced, the complex peptide mixture can be analyzed by RP-HPLC; however, only the level of total clipping is identified. Since RC-CZE separation relies upon differences in molecular charge/size ratio, it can resolve the three rhPDGF-BB forms differing in the additional exposed residues. The choice of reversed-charge CZE columns (amine-coated column) allows proteins of high pI such as rhPDGF-BB (pI > 10) to be readily analyzed while minimizing protein loss from column adsorption. To simplify the electropherogram of clipped forms, the sample is treated first with carboxypeptidase B to reduce the charge microheterogeneity of partial Arg32 truncation. Analysis of rhPDGF-BB by RC-CZE yields a baseline separation between the three forms, intact and single- and double-clipped rhPDGF-BB.  相似文献   

6.
The binding properties of codeine, morphine (as representative opium alkaloids), and methadone (a synthetic pharmacologically similar compound) were studied with selected human serum proteins. The methodology involved equilibrium and dynamic dialysis using 3H-and/or 14C-labeled compounds. For estimation of the percent binding with equilibrium dialysis, concentrations of the ligand used were approximately therapeutic blood levels and another concentration 30-60 times higher. The percent binding to whole human serum ranged from about 20% for morphine to almost 60% for methadone. Of the human serum proteins investigated, the highest percent binding was found with albumin, except for methadone for which it was beta-globulin III. The affinity for other serum proteins varied with the ligand. In studies with albumin using dynamic dialysis, the plots of nubar divided by free concentration versus nubar were similar for all three ligands studied and had positive slopes, unlike those reported for acidic compounds for which the slope is always negative. In studies of binding of one ligand in the presence of another, significant competition was demonstrated, suggesting that the same binding sites were involved.  相似文献   

7.
An automated free-solution isotachophoresis system (FS-ITP) for preparative fractionation of biopolymers is described, operated in a batch mode. The dimension of the separation chamber allows an up to 1200-fold higher sample load compared to separation in capillaries of 180 microm inner diameter as used in analytical capillary isotachophoresis (C-ITP). The preparative capacity of the system is within the milligram range. The method is fully compatible with analytical C-ITP, which is essential for preparative-scale isotachophoresis with regard to optimization of electrolyte systems and the search for suitable spacers. As a model application the fractionation of human serum proteins is reported. The collected fractions were analyzed by C-ITP and agarose gel electrophoresis.  相似文献   

8.
Weak biospecific recognition has been established for affinity separation in high performance liquid chromatography (HPLC). The use of weak affinity chromatography (WAC) has been limited previously by the insufficient separation efficiency achieved, allowing only some 1000 plates/m to be obtained. However, it has been shown that chiral drug separation can be performed with capillary affinity gel electrophoresis (CAGE) at considerably improved efficiency as compared with traditional chromatographic procedures. Our present study demonstrates the potential of weak affinity monoclonal antibodies as a generic method for immunologically based separations in capillary electrophoresis. Monoclonal antibodies were polymerized within a silica capillary and were used for the separation of structurally similar carbohydrate antigens. The results indicate that weak biospecific interactions can be utilized in a CAGE format to produce highly selective separation of the alpha- and beta-forms of p-nitrophenyl-labeled maltose. It remains to be seen, however, how efficient weak affinity separation in CAGE can be compared with affinity HPLC protocols. Details of typical separations and of the preparation of the antibody gel are presented.  相似文献   

9.
Affinity capillary electrophoresis is a new method for studies of biomolecular recognition. Applications reported in the literature include chiral separation of racemic biomolecules, measurement of binding constants, estimation of kinetic on- and off-rate constants, determination of binding stoichiometries (a useful tool in examining electrostatic interactions), estimation of effective charges and molecular weights of proteins, characterization of enzymatic activities and library screening for tight-binding drug candidates in solution. This technique demands only small amounts of sample (nanolitre injection volumes, picograms of proteins), involves no radiolabelled materials or chemically immobilized ligands, and does not require changes in spectroscopic characteristics upon binding. This paper reviews the most recent applications of affinity capillary electrophoresis and its use in the analysis of biomolecules.  相似文献   

10.
To determine the forms of cardiac troponin I (cTnI) circulating in the bloodstream of patients with acute myocardial infarction (AMI) and patients receiving a cardioplegia during heart surgery, we developed three immunoenzymatic sandwich assays. The first assay involves the combination of two monoclonal antibodies (mAbs) specific for human cTnI. The second assay involves the combination of a mAb specific for troponin C (TnC) and an anti-cTnI mAb. The third assay was a combination of a mAb specific for human cardiac troponin T (cTnT) and an anti-cTnI mAb. Fifteen serum samples from patients with AMI, 10 serum samples from patients receiving crystalloid cardioplegia during heart surgery, and 10 serum samples from patients receiving cold blood cardioplegia during heart surgery were assayed by the three two-site immunoassays. We confirmed that cTnI circulates not only in free form but also complexed with the other troponin components (TnC and cTnT). We showed that the predominant form in blood is the cTnI-TnC binary complex (IC). Free cTnI, the cTnI-cTnT binary complex, and the cTnT-cTnI-TnC ternary complex were seldom present, and when present, were in small quantities compared with the binary complex IC. Similar results were obtained in both patient populations studied. These observations are essential for the development of new immunoassays with improved clinical sensitivity and for the selection of an appropriate cTnI primary calibrator.  相似文献   

11.
This study compared the diagnostic accuracy of the measurement of serum cardiac troponin I (cTnI) with creatine kinase (CK) MB mass in patients with minor myocardial injury whose measured total CK activity did not exceed twice the upper reference limit (300 U/L for men; 200 U/L for women). Forty-eight consecutive patients presenting with chest pain and with in-hospital documentation of myocardial injury were enrolled. Electrocardiogram, echocardiogram, and serial serum CK-MB mass, cTnI, and total CK were measured over 36 h after admission. Peak total CK activity was within normal limits in 28 patients (58%). The mean (+/- SD) peak CK-MB mass and cTnI concentrations were: 16.4 (11.8) micrograms/L and 132 (13.0) micrograms/L; respectively. The peak biochemical marker index (defined as CK-MB or cTnI divided by its respective upper reference limit) was significantly (P < 0.05) higher for cTnI than for CK-MB from 7 to 36 h. The clinical sensitivity for detection of myocardial injury for cTnI was 100% [95% confidence interval (CI): 87.2% to 100%], compared with 81.8% (CI: 67.3% to 91.8%) for CK-MB. Thus, cTnI was more sensitive than CK-MB mass for detection of myocardial injury in patients with small increases of total CK.  相似文献   

12.
BACKGROUND: In the failing human heart myofibrillar calcium sensitivity of tension development is greater and maximal myofibrillar ATPase activity is less than in the normal heart. Phosphorylation of the cardiac troponin I (cTnI)-specific NH2-terminus decreases myofilament sensitivity to calcium, while phosphorylation of other cTnI sites decreases maximal myofibrillar ATPase activity. METHODS AND RESULTS: We examined cTnI phosphorylation in left ventricular myocardium collected from failing hearts at the time of transplant (n=20) and normal hearts from trauma victims (n=24). The relative amounts of actin, tropomyosin, and TnI did not differ between failing and normal myocardium. Using Western blot analysis with a monoclonal antibody (MAb) that recognizes the striated muscle TnI isoforms, we confirmed that the adult human heart expresses only cTnI. A cTnI-specific MAb recognized two bands of cTnI, designated cTnI1 and cTnI2, while a MAb whose epitope is located in the cTnI-specific NH2-terminus recognized only cTnI1. Alkaline phosphatase decreased the relative amount of cTnl1, while protein kinase A and protein kinase C increased cTnI1. The percentage of cTnI made up of cTnI1, the phosphorylated form of TnI, is greater in the normal than the failing human heart (P<.00). CONCLUSIONS: This phosphorylation difference could underlie the reported greater myofibrillar calcium sensitivity of failing myocardium. The functional consequence of this difference may be an adaptive or maladaptive response to the lower and longer calcium concentration transient of the failing heart, eg, enhancing force development or producing ventricular diastolic dysfunction.  相似文献   

13.
The IgG transporter responsible for ferrying maternal IgG across the human placenta to fetal circulation has not been identified, although the human homologue of the neonatal rat Fc receptor (FcRn), a heterodimer with pH-dependent IgG affinity, structurally similar to MHC Class I molecules, was recently proposed as a candidate. Affirming this hypothesis, we describe herein the specific copurification from human placenta of 46- and 14-kDa proteins by IgG affinity at acid pH. The larger protein, characterized by its amino acid sequence and by immunoblot, is the alpha-chain of human FcRn (hFcRn). The smaller is beta2-microglobulin. Their coisolation by ligand affinity suggests that they comprise the hFcRn heterodimer. Placenta sections stained immunohistochemically with anti-hFcRn alpha-chain peptide Abs show extensive expression of hFcRn in the syncytiotrophoblast and traces in the endothelium and other unidentified cells of the villus stroma. We find alpha-chain mRNA by Northern analysis in human placenta and in human trophoblast-like cell lines (JEG-3, ED27) but not in a human myelocytic cell line (HL60). We suggest that the placental hFcRn heterodimer may transport IgG to the fetus by a mechanism in which maternal IgG is pinocytosed nonspecifically and then carried to fetal tissues by a pH gradient from acidic endosomes to the pH-neutral basolateral surface of the syncytiotrophoblast. Furthermore, the known characteristics of FcRn suggest a wider function, that it is the receptor postulated by Brambell in the 1960s to regulate tissue and serum IgG concentrations by controlling IgG transport and catabolism.  相似文献   

14.
Four phosphorylation degrees of cardiac troponin I (cTnI) have been characterized, namely, a dephospho, a bisphospho, and two monophospho states. Here we describe for the first time a role of the monophosphorylated forms. We have investigated the interaction between the cardiac troponin subunits dependent on the phosphorylation state of cTnI by surface plasmon resonance (SPR) spectroscopy. The monophosphorylated forms were generated by mutating each of the two serine residues, located in human cTnI at positions 22 and 23, to alanine. Association and dissociation rate constants of binary (cTnI-cTnT and cTnI-cTnC) and ternary (cTnI/cTnC complex-cTnT) complexes were determined. Mono- and consecutive bisphosphorylation of cTnI gradually reduces the affinity to cTnC and cTnT by lowering the association rate constants; the dissociation rate constants remain unchanged. Phosphorylation also affects formation of the ternary complexes; however, in this instance, association rate constants are constant, and dissociation rate constants are enhanced. A model of cardiac troponin is presented describing an induction of distinct conformational changes by mono- and bisphosphorylation of cTnI.  相似文献   

15.
A method for the identification of proteins by their amino acid sequence at the low-femtomole to subfemtomole sensitivity level is described. It is based on an integrated system consisting of a capillary zone electrophoresis (CZE) instrument coupled to an electrospray ionization triple- quadrupole tandem mass spectrometer (ESI-MS/MS) via a microspray interface. The method consists of proteolytic fragmentation of a protein, peptide separation by CZE, analysis of separated peptides by ESI-MS/MS, and identification of the protein by correlation of the collision-induced dissociation (CID) patterns of selected peptides with the CID patterns predicted from all the isobaric peptides in a sequence database. Using standard peptides applied to a 20-microns-i.d. capillary, we demonstrate an ESI-MS limit of detection of less than 300 amol and CID spectra suitable for searching sequence databases obtained with 600 amol of sample applied to the capillary. Successful protein identification by the method was demonstrated by applying 50 and 38 fmol of a tryptic digest of the proteins beta-lactoglobulin and bovine serum albumin, respectively, to the system.  相似文献   

16.
Current markers of myocardial injury lack specificity in patients with end-stage renal disease (ESRD). In particular, a false positive creatine kinase-MB (CKMB) elevation occurs in 5-10% of patients with ESRD. The aim of this study was to ascertain the relationship between CKMB and cardiac troponin I (cTnI), a new, highly sensitive and specific marker for myocardial injury, in the authors' dialysis population and compare their specificities. Blood samples were obtained from 112 dialysis patients (35 in peritoneal dialysis; 77 in hemodialysis). Patients were asymptomatic for cardiac ischemia and skeletal muscle injury. Mean +/- SD CKMB mass was 3.16 +/- 2.26 microg/L (range, 0.34-13.62), and cTnI was 0.025 +/- 0.061 ng/ml (range, 0.001-0.496). CKMB and cTnI levels did not correlate (r2 = 0.002; p = 0.61). CKMB mass concentration was significantly higher in men and in diabetics. No patient had a cTnI level greater than 1.5 microg/L, and eight asymptomatic patients had a CKMB mass greater than 6.7 microg/L. These data suggest a specificity of 100% for cTnI vs 94.6% for CKMB at these cutoff values. It is suggested that cTnI replace CKMB as a marker of myocardial injury in patients with ESRD.  相似文献   

17.
Serial plasma concentrations of myoglobin, creatine kinase MB (CK-MB) isoenzyme, and cardiac troponin I (cTnI) were measured in 25 patients with a confirmed diagnosis of acute myocardial infarction (AMI), and 74 patients who were suspected of AMI but were subsequently ruled out for this diagnosis. The cutoff concentration for the cTnI assay was optimally determined to be 2.5 ng/mL. Of the three markers, myoglobin had the highest clinical sensitivity (50 percent) when blood was collected between 0 to 6 h after the onset of chest pain. Assays for all serum markers used had high clinical sensitivity (> 93 percent) 6 to 24 h after onset. The CK-MB remained highly sensitive for 48 h, while cTnI was sensitive for up to 72 h. Between 72 and 150 h, cTnI had a clinical sensitivity of 70 percent as compared to 21 percent and 18 percent for myoglobin and CK-MB, respectively. The clinical specificity of cTnI for non-AMI patients was equivalent to CK-MB and significantly higher than for myoglobin. The clinical efficiency of cTnI for all samples was better than either CK-MB or myoglobin, owing mainly to the wider diagnostic window. The specificity of cTnI for 59 patients with chronic renal failure, skeletal muscle trauma and disease was better than all of these markers including cardiac troponin T (cTnT). Results of this study show that cTnI is an effective marker for the retrospective diagnosis of AMI, and consideration should be given to its use in place of CK-MB.  相似文献   

18.
Pentraxins are a family of pentameric serum proteins that have been conserved in evolution and share sequence homology, similar subunit assembly and the capacity for calcium-dependent ligand binding. The classical pentraxins are human C-reactive protein (CRP) and serum amyloid P component (SAP). The sequence homology and gene organization indicate that they arose from a gene duplication of an ancestral pentraxin gene. They are usually isolated based on their affinity for phosphorylcholine and agarose, respectively. We have used this method for isolation of pentraxin-like proteins from normal serum of Atlantic salmon (Salmo salar), common wolffish (Anarhichas lupus), cod (Gadus morhua) and halibut (Hippoglossus hippoglossus). Although pentraxin structures have not been verified, the isolated proteins all appear to be pentraxin-like based on their binding specificity, molecular weight of subunits, cross-reactivity with antibodies to human pentraxins and N-terminal amino acid sequences. However, with the described method only one pentraxin-like protein was detected in each of the fish species.  相似文献   

19.
The phenomenon of differential charge distribution on sperm surface membrane has been utilised here in a low e.m.f. (electro motive force) capillary electrophoresis system to effect separation of sperm heads from disintegrated mixed spermatozoal subfractions. Washed caudal sperm of goat (Black Bengal variety) and ejaculated washed human sperm were fractionated by sonication into head, mid-piece and tail portions. Routine techniques of density gradient centrifugation on Percoll and/or sucrose were performed with sonicated spermatozoa for separation into their respective subfractions. The products obtained were not free of contamination in either case. Mixed sperm fractions when subjected to the afore mentioned modified capillary electrophoresis technique only the head pieces exhibited high affinity for migration towards the cathode terminal. With this method around 50% of the total sperm heads were separated and collected in absolutely pure form at the cathode side within 2 hrs. at 150 volts (V) and 1.5 milliampere (mA) current at 37.5 degrees C. A 4 cm. long capillary tube with a bore size 1.2 mm. was used for this purpose.  相似文献   

20.
We have analyzed by different immunological methods the proteolytic degradation of cardiac troponin I (cTnI) in human necrotic tissue and in serum. cTnI is susceptible to proteolysis, and its degradation leads to the appearance of a wide diversity of proteolytic peptides with different stabilities. N- and C-terminal regions were rapidly cleaved by proteases, whereas the fragment located between residues 30 and 110 demonstrated substantially higher stability, possibly because of its protection by TnC. We conclude that antibodies selected for cTnI sandwich immunoassays should preferentially recognize epitopes located in the region resistant to proteolysis. Such an approach can be helpful for a much needed standardization of cTnI immunoassays and can improve the sensitivity and reproducibility of cTnI assays.  相似文献   

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