Fragmentation and polymeric complexes of albumin in human urine

Analysis of urine proteins of some individuals with proteinuria by SDS-PAGE and silver staining revealed protein bands in urine which did not appear to be present in plasma. The bands migrated with apparent molecular weights of 260 000, 180 000, 110 000, 45 000, 40 000, 30 000, 24 000, 18 000 and 11 000. These bands were shown to be albumin polymer and fragments by using a polyclonal antibody to (a) immunoprecipitate radiolabelled urine proteins, and (b) identify bands blotted from SDS-PAGE gels onto nitrocellulose paper. The specificity of the polyclonal anti-albumin antibody was confirmed by using two mouse monoclonal antibodies raised against human albumin which, between them, recognized the same protein bands on nitrocellulose paper as did the polyclonal antibody. The results of these studies of albumin in human urine confirm that albumin exists as polymer and also show that albumin fragmentation occurs in urine. Fragmentation occurs by proteolysis of the albumin molecule both at sites within and outside disulfide loops. The predominant cleavage site appears to be approximately two-fifths of the distance from one end of the albumin molecule to produce disulfide-linked fragments of about 45 000 and 30 000 molecular weight.     

A map of urine proteins based on one-dimensional SDS-polyacrylamide gel electrophoresis and Western blotting using one microliter of unconcentrated urine

A sensitive one-dimensional SDS-polyacrylamide gel electrophoretic system was devised whereby the proteins in 1 microliter of unconcentrated urine could be visualized by silver staining over the range 9,000-900,000 molecular weight. Identification of urine proteins was confirmed by Western blotting using peroxidase labelled antibodies. A map of the major proteins visualized in urine from individuals with renal disease was constructed. We conclude that the information available from the simple analysis of proteins according to their size is limited to general conclusions regarding whether proteinuria is likely to be of tubular or glomerular or mixed origin. More specific identification of individual proteins is not feasible because simple protein staining is not sufficiently reliable to identify individual proteins. The reasons for this conclusion are as follows: many proteins in urine migrate with similar apparent molecular weights, some proteins are not visualized by silver staining, and albumin polymeric complexes and fragments can be present at almost any molecular weight. However, one-dimensional SDS-polyacrylamide gel electrophoresis together with Western blotting does provide reliable information which might be clinically and experimentally useful.     

Characterization of immunochemically nonreactive urinary albumin

BACKGROUND: Conventional immunoassays underestimate the urinary albumin concentration because intact albumin in urine exists in two forms, immunoreactive and immunochemically nonreactive. METHODS: Urinary albumin concentration measured by HPLC (which measures total albumin, i.e., the sum of immunoreactive albumin + immunochemically nonreactive albumin) or RIA was compared with densitometric analysis of albumin bands in diabetic urine samples separated by either native polyacrylamide gel electrophoresis (PAGE) or reducing sodium dodecyl sulfate (SDS)-PAGE. Immunochemically nonreactive albumin was also isolated from diabetic urine (relative amount detected, 70-80% of the expected) and was tested for contamination by common urinary proteins by native PAGE, ELISA, and capillary electrophoresis. RESULTS: Urinary albumin concentrations measured by native PAGE and HPLC were better correlated (r(2) = 0.83) than concentrations measured by native PAGE and RIA (r(2) = 0.62) because under native conditions both native PAGE and HPLC detect total albumin and not only the immunoreactive albumin alone that is measured by RIA. Urinary albumin concentrations measured by reducing SDS-PAGE and RIA were better correlated (r(2) = 0.84) than concentrations measured by reducing SDS-PAGE and HPLC (r(2) = 0.65) because under reducing conditions immunochemically nonreactive albumin is unstable and fragments into many smaller peptides. The partially purified preparation was found to contain <1% contamination by common urinary proteins and is stable to freezing and frequent freeze/thaw cycles. CONCLUSIONS: The results are consistent with the interpretation that immunochemically nonreactive albumin has a limited number of polypeptide chain scissions and is held together by noncovalent intrachain bonding and disulfide bonds. Detection of this molecule is likely to be of clinical importance in diagnosing kidney disease as well as cardiovascular disease.     

Distinction between epidermal antigens binding pemphigus vulgaris and pemphigus foliaceus autoantibodies.

Pemphigus vulgaris (PV) and pemphigus foliaceus (PF) are autoimmune blistering diseases in which antibodies develop to the cell surface of epidermal cells. In this study we sought to determine the antigenic specificity of antibodies in the sera of patients with PV and PF. Sera from 12 patients with PV were used to immunoprecipitate extracts of cultured human epidermal cells that were radiolabeled with 14C-amino acids. Immunoprecipitates were identified by SDS polyacrylamide gel electrophoresis (PAGE) and fluorography. All 12 PV sera precipitated a protein which, when reduced, displayed chains of 130,000 and 80,000 mol wt on SDS-PAGE. Electrophoresis under nonreducing conditions identified a 210,000-mol wt molecule, which was presumably formed by disulfide crosslinking of the 130,000 and 80,000-mol wt chains. Immunoprecipitates of epidermal cell extracts that were labeled with 14C-glucosamine indicated that the 130,000-mol wt chain. Seven of eight PF sera, which were run concurrently with the PV sera in this immunoprecipitation assay, did not precipitate this glycoprotein, nor did they specifically precipitate any protein. To determine if a specific molecule which reacted with antibodies in PF sera could be identified, we used immunoblot analysis of extracts of normal human epidermis. The proteins in these extracts were reduced, separated by SDS-PAGE, and electrophoretically transferred to nitrocellulose sheets or to 2-aminophenylthioether paper. Immunoperoxidase staining of the transferred proteins with PF sera indicated that four of eight PF sera contained antibodies that stained a protein band of 160,000 mol wt. Indirect immunofluorescence, using normal human skin as the substrate, indicated that IgG that was eluted from this protein band stained the epidermis in a cell surface pattern. PV sera did not specifically recognize any bands by immunoblot analysis. Immunoblots performed with PV antigen that was immunoprecipitated from cell culture extracts suggested that, once denatured for SDS-PAGE, PV antigen is no longer immunoreactive. Taken together, these data indicate that: autoantibodies contained in PV sera from various patients have a unique molecular specificity; autoantibodies from most PF sera have a specificity different from that of PV autoantibodies; and autoantibodies from various PF patients may not have identical antigenic specificities. These differences in antigenic specificity between PV and PF sera may account for the clinical and histologic differences between these diseases.     

Rapid differentiation of glomerular and tubular proteinuria by sodium dodecyl sulfate polyacrylamide gel electrophoresis

Serum and urine protein samples were incubated with sodium dodecyl sulfate and subjected to polyacrylamide gel electrophoresis. There was a linear relationship between the logarithm of the molecular weight of the protein and its distance of migration (R_F) on the gel. This relationship held over a molecular weight range from at least 160000 to 12000, i.e., over a range particularly useful for serum proteins and urine proteins. This system was then applied to the analysis of both serum and urine proteins. Glomerular proteinuria (characterized by the presence of albumin and plasma proteins of a molecular size larger than albumin) and tubular proteinuria (characterized by the presence of albumin and proteins of molecular size smaller than albumin) were readily distinguished. The results were similar to those obtained on the same samples with Sephadex G-200 and Sephadex G-75 columns. The SDS polyacrylamide gel electrophoretic method resolved the proteins of varying molecular weights better than did Sephadex. It was simple to use, and very much more rapid; thus, it was suitable for the examination of large numbers of samples.     

Uromucoid (Tamm-Horsfall glycoprotein) forms different polymeric arrangements on a filter surface under different physicochemical conditions

Normal human urine cannot be forced through a 0.2 micron filter. To investigate the reason for this phenomenon, uromucoid (Tamm-Horsfall protein) was purified from human urine and its capacity to block a 0.2 micron Millipore filter was measured under different conditions. In the presence of cations (H+, Na+, Ca2+) uromucoid blocked the filter. The blocking varied with cation concentration. Scanning electron microscopy of the filter surface revealed different arrangements of polymerized uromucoid coating the filter surface depending on ionic conditions. In the presence of 100 mmol/l NaCl or 1 mmol/l CaCl2 uromucoid polymers were present in a fibrous arrangement. In the presence of both NaCl and CaCl2 a dense mat of uromucoid polymers was present together with clumps of aggregated polymer. In the absence of ions uromucoid formed a homogeneous coat on the filter surface (as demonstrated by scanning electron microscopy, Western blotting and 125I-uromucoid binding studies) but did not block the filter. Similar fibrous and highly aggregated arrangements of uromucoid polymer were seen in hyaline casts from urine. These data are consistent with the concept that the uromucoid glycoprotein can exist in several different polymeric forms under different ionic conditions.     

Human autoantibodies against a desmosomal core protein in pemphigus foliaceus

Pemphigus foliaceus (PF) is a human autoimmune disease in which antibodies are directed against the cell surface of epidermal cells with resultant blister formation. The histopathology of these blisters indicates that cells have detached from each other, and electron microscopy of early blisters shows diminished numbers, to complete loss, of desmosomes as well as abnormalities of the tonofilament- desmosome complex. In this study we demonstrate that autoantibodies from certain PF patients bind to a desmosomal core glycoprotein called desmoglein (DG) I. Proteins in extracts of normal human epidermis were separated by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE), then transferred to nitrocellulose or 2- aminophenylthioether paper for immunoperoxidase staining. Results of these immunoblots indicated that sera from 6 of 13 PF patients specifically and intensely stained an approximately 160,000 mol wt polypeptide, "PF antigen". Such staining was not seen with normal human sera or sera from patients with pemphigus vulgaris or bullous pemphigoid, two autoimmune blistering skin diseases that are clinically, histologically, and immunochemically distinct from PF. However, rabbit antiserum directed against DGI, that was isolated from bovine muzzle desmosomes, stained a polypeptide band which co-migrated with PF antigen. Furthermore, when proteins from extracts of normal human epidermis were electrophoresed in two dimensions (isoelectric focusing, then SDS-PAGE) before transfer to nitrocellulose for immunoperoxidase staining, PF antibodies and antibodies to DGI stained identical spots. Finally, PF sera as well as PF IgG that was affinity purified with PF antigen from normal human epidermis, both selectively bound to DGI extracted from bovine muzzle desmosomes. These studies demonstrate that the human autoantibodies from certain patients with PF, a disease of epidermal cell adhesion, are directed against a desmosomal core protein.     

Correlation between the cystic fibrosis protein detected by isoelectric focusing and a protein of 12 kDa detected by SDS-polyacrylamide gel electrophoresis

We have studied both by isoelectric focusing and polyacrylamide gel electrophoresis in the presence of SDS, sera of individuals homozygous (25) and heterozygous (26) for cystic fibrosis and compared them to controls (13). As in our first study [1], the protein with a pI value of 8.4 called 'cystic fibrosis protein' or CFP, was found in about 70% of homozygous and carriers and in 15% of controls. When the same sera were analysed by polyacrylamide gel electrophoresis in the presence of SDS and after staining with Coomassie Blue, an additional protein with a molecular weight close to 12,000 (P12) was present in most of the sera containing CFP, suggesting a close relationship between the two proteins. By increasing the sensitivity of staining by using silver nitrate, P12 was detected with variable intensity in almost all sera of homozygotes and heterozygotes and at the level of traces in all normal sera. These data suggest that P12, like CFP, would be a normal serum protein quantitatively increased in affected subjects.     

Missing band 7 membrane protein in two patients with high Na, low K erythrocytes.

We investigated the erythrocyte membrane proteins of two patients with congenital hemolytic anemia due to increased permeability of the erythrocyte membrane to Na and K (hereditary stomatocytosis and cryohydrocytosis). One-dimensional sodium dodecyl sulfate (SDS) gel electrophoresis resolved the band 7 erythrocyte membrane proteins into three components with approximate molecular weights of 30,000, 28,000, and 26,000. The 28,000-dalton component was decreased in both patients with permeability disorders. Two-dimensional electrophoresis (nonequilibrium pH gradient electrophoresis in the first dimension combined with SDS gel electrophoresis in the first dimension combined with SDS gel electrophoresis in the second dimension) resolved the 28,000-dalton component from normal erythrocyte membranes into two proteins with different isoelectric points, designated 22 x 8 and 60 x 8. In the patients with hereditary stomatocytosis and cryohydrocytosis, 22 x 8 was completely absent, whereas 60 x 8 was detected as usual. In contrast, all the band 7 proteins (including 22 x 8) were invariably present in a survey of normal subjects and reticulocytosis controls. The unique finding of a missing band 7 protein in the patients with hereditary stomatocytosis and cryohydrocytosis raises the possibility that the absence of this protein is responsible for the increased Na and K permeability in these disorders.     

Presence of immunounreactive albumin in the urine of diabetic patients

Recent studies have demonstrated that conventional immunochemical assays underestimate urinary albumin concentration because of the presence of immunounreactive albumin. It has been reported that intact urinary albumin in 24-hr diabetic urine samples could be detected as total concentration (immunoreactive+immunounreactive) by an HPLC method based on size exclusion chromatography. The aim of this study was to investigate urinary albumin concentration in diabetic spot urine samples by comparing the HPLC method with several other methods. The albumin concentrations on 80 diabetic spot urine specimens were measured by turbidimetric immunoassay (TIA), high performance liquid chromatography (HPLC), and a dipstick method.In addition, they were also analyzed by reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) and native polyacrylamide gel electrophoresis (Native PAGE). The albumin concentrations derived from diabetic spot urine samples measured by the HPLC method were higher than those of the other methods except for five of 80 samples. Furthermore, the albumin concentrations analyzed by Native PAGE were higher than SDS PAGE in 61 (76.2%) of 80 samples. This study suggests the need for evaluating diabetes not only by HPLC, but also by combining it with another method.     

乙酰胆碱受体的初步纯化及其抗体测定方法的建立

用Ｔｒｉｔｏｎｘ－１００提取，抗人全血清－Ｓｅｐｈａｒｏｓｅ４Ｂ柱免疫吸附和ＳｅｐｈａｄｅｘＧ－１５０凝胶过滤的方法，自人骨骼肌提取得乙酰胆碱受体（ＡｃｈＲ），ＳＤＳ－ＰＡＧＥ呈一条４４ｋＤ的主带，用此ＡｃｈＲ作包被抗原，建立了检测ＡｃｈＲ－Ａｂ的间接ＥＬＩＳＡ法。若以对照组吸光度均值加２倍标准差为正常上限，则重症肌无力（ＭＧ）患者中，５０％抗ＡｃｈＲ自身抗体（ＡｃｈＲ－Ａｂ）阳性，其他自身免疫病（ＳＬＥ，ＲＡ等）者检出率极低。     

Variability of standard clinical protein assays in the analysis of a model urine solution of fragmented albumin

OBJECTIVES: This study investigates the sensitivity of various standard clinical techniques in the detection of albumin fragments. The significance of this work is in the detection of urinary proteins, such as albumin, which has recently been discovered to be excreted as mainly peptide fragments as a result of filtered albumin undergoing degradation during renal passage. All filtered proteins undergo a similar degradation process. DESIGN AND METHODS: Albumin digested with trypsin was used as a model urine solution. The solution was assayed for albumin concentration by various methods including the biuret assay that is known to detect urinary albumin fragments. The digest solution was also analyzed by various clinically used chromagen assays, electrophoretic and chromatographic methods to determine whether they are able to detect the fragmented protein. RESULTS: The benzethonium chloride, Coomassie blue, and pyrogallol red assays for urine protein, the immunoassay for human albumin and sodium dodecyl sulfate polyacrylamide gel electrophoresis with Coomassie blue staining were unable to detect the albumin fragments. Capillary electrophoresis was sensitive to the fragments but with low resolution. High-performance liquid chromatography gave the best results. CONCLUSIONS: Many techniques utilized to assay patient urine samples are unable to detect fragmented albumin and, hence, will severely underestimate albumin and protein excretion.     

93例肾脏疾病患者SDS-AGE非浓缩尿蛋白电泳分析

目的观察十二烷基硫酸钠-琼脂糖凝胶(SDS-AGE)非浓缩尿蛋白电泳在肾脏疾病诊断中的价值。方法采用SDS-AGE非浓缩尿蛋白电泳技术对93例肾脏疾病患者的蛋白尿进行分析。结果 93例肾病患者尿蛋白电泳中,肾小球性蛋白尿50例,肾小管性蛋白尿6例,混合型蛋白尿9例,白蛋白尿24例。结论 SDS-AGE非浓缩尿蛋白电泳的检测对明确肾病患者尿蛋白的性质、来源及各种蛋白的含量,判断肾脏损伤类型及程度具有较高的临床应用价值,且取材简便,敏感性高。     

应用尿液蛋白质组技术筛选多发性骨髓瘤相关蛋白的初步研究

目的应用蛋白质组学相关技术筛选多发性骨髓瘤（MM）患者尿液中差异表达的蛋白质,寻找与MM诊断相关的疾病标志物。方法收集MM组、疾病对照组（肾脏疾病）、健康对照组尿液各8例,TCA沉淀法提取尿液蛋白质后建立双向凝胶电泳图谱;Image Master软件筛选差异蛋白质;利用MALDI-TOF-MS获得肽质量指纹图谱,通过Mas-cot软件进行数据库搜索,得到蛋白质鉴定结果。结果筛选出57个差异蛋白点,43个在MM组上调,14个下调。对其中30个差异蛋白点进行分析,鉴定出甘露聚糖结合凝集素相关蛋白19（Map19）、人组织相容性抗原A2（Hla-A2）、视黄醇结合蛋白（RBP）、转甲状腺素蛋白（TTR）、免疫球蛋白轻链、α2-糖蛋白1（ZAG1）等9类差异表达的蛋白质。结论应用尿液蛋白质组技术成功筛选鉴定出MM尿液中存在的差异蛋白质,可能是MM疾病诊断相关标志物,尚需要进一步验证。     

The purification and partial characterization of an insulin-like protein from human serum.

A preparative scheme has been developed for the purification of a trace protein in human serum exhibiting nonsuppressible insulin-like activity (NSILA). This scheme consisted of (a) adsorption chromatography of serum utilizing the sulfonic acid polystyrene resin, Dowex 50, at pH 6.8; (b) (200 gel filtration at pH 8.9; and (c) acrylamide gel electrophoresis in a discontinuous preparative system. Throughout all procedures, NSILA fractionated as a single molecular species approximating 90,000 mol wt. The purified protein exhibited a single band by disk gel electrophoresis, an isoelectric pH approximating 6.2, doublet bands of 90,000 mol mt by analytical sodium dodecyl sulfate gel electrophoresis, and a biologic specific activity approximating 50 mU/mg. Serum somatomedin (sulfation factor) activtiy did not fractionate with NSILA in this scheme, and partially purified NSILA did not stimulate radiosulfate uptake into hypophysectomized rat costal cartilage. This protein appears to represent the major constituent of serum NSILA: its purification and partial characterization provides the first step towards elucidation of its metabolic role.     

Uromucoid in normal urine

Uromucoid was determined in urine from persons or various ages with normal kidney function by a radial immunodiffusion technique. Uromucoid concentration was inversely correlated to diuresis. Excretion rates showed a weak correlation to body surface area in young adults of both sexes. Children had a greater excretion of uromucoid per m2 than young adults, and even newborn had a considerable excretion. Elderly subjects had similar excretion rates to young adults. Excretion rates were uninfluenced by sex, salt load or diuresis, and no difference between day and night excretion rates was observed. Intraindividual fluctuations were small, and determination of uromucoid in one single night's urine therefore gives an adequate expression of the uromucoid excretion rate.     

Sweat protein components tested by SDS-polyacrylamide gel electrophoresis followed by immunoblotting

Sweat samples were collected in a sauna from 74 healthy volunteers (72 men and 2 women) and concentrated. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of the individual samples revealed, in general, five main proteins and four PAS positive components. In pooled sweat, a method of SDS-PAGE followed by immunoblotting with specific antisera or antibodies against 24 human serum components was applied, and three out of the five main proteins showed the same molecular weights and antigenicities corresponding to serum albumin (67,000 Da), Zn-alpha 2-glycoprotein (42,000 Da) and lysozyme (14,000 Da). Moreover, orosomucoid, transferrin, IgG and IgA were demonstrated in the pooled sweat. Although alpha 1-antitrypsin was probably in the pooled sweat, other serum components could not be detected. On the pooled and individual sweat samples, anti-carcinoembryonic antigen (CEA) formed three bands at 42,000, 19,000 and 18,000 Da, but the antibody did not react with normal serum. It might be considered from these molecular weights that those sweat components are CEA-related antigens.     

Simple method for quantification of Bence Jones proteins

BACKGROUND: Quantification of free monoclonal light chains in urine [Bence Jones proteins (BJPs)] is used to diagnose multiple myeloma and to evaluate response to treatment. We have developed and evaluated an optimized approach for quantification of BJPs. METHODS: High-resolution gel electrophoresis of unconcentrated urine and albumin calibrators was carried out on Sebia's Hydrasys instrument with Hydragel HR agarose gels. After staining with acid violet, the gels were scanned densitometrically. The staining intensities of BJP bands relative to the staining intensities of albumin solutions were used to determine the BJP concentrations. Results for patient samples were compared with conventional agarose gel electrophoresis on concentrated samples. RESULTS: The relationships between staining intensity and the protein concentrations of albumin and BJPs were linear up to protein concentrations of approximately 2000 mg/L. The detection limit was approximately 20 mg/L. The interassay imprecision (CV) was approximately 8% (n = 23, duplicate analysis), and the results (y) showed a close positive relationship to the comparison method: slope = 0.82 (confidence interval, 0.75-0.88); y-intercept = 34 (-14 to 81) mg/L; n = 29; r(2) = 0.96. CONCLUSIONS: Agarose gel electrophoresis of unconcentrated urine samples together with a series of albumin calibrators followed by acid violet staining and densitometric scanning is sufficiently reproducible and sensitive to quantify clinically relevant BJPs.     

Purification of macrophage deactivating factor

Macrophage deactivation factor (MDF) in P815 tumor cell-conditioned medium was assayed by its suppression of the ability of activated mouse peritoneal macrophages to release hydrogen peroxide. MDF displayed properties of a soluble protein(s) associated with both low (8-25,000) and high (greater than 450,000) Mr fractions. MDF was purified 6,140-fold by a seven-step procedure: extraction with acid-ethanol; precipitation with ether; and fractionation on gel filtration, anion-exchange, diphenyl reversed-phase and C4 reversed-phase HPLC columns, the last column twice. The final preparation contained two species: (a) a approximately 13,000 Mr band on reducing or nonreducing SDS-PAGE and on autoradiograms after radioiodination with chloramine T, and (b) a 66,000 Mr species ranging from approximately 5% to approximately 50% of the protein detectable by silver strain. The 66,000 Mr species was identified as albumin from its NH2-terminal amino acid sequence. However, no amino acid sequence could be obtained for the approximately 13,000 Mr species, either in fluid phase or after electroelution of the corresponding SDS-PAGE band. Thus, approximately 13,000 Mr MDF associates tightly with albumin through a variety of separation techniques, and may have a blocked NH2 terminus. Purified MDF afforded 50% inhibition of activated macrophage H2O2 releasing capacity at a concentration of 1-10 nM. Separation of MDF from most higher Mr moieties was associated with disproportionately small increases in specific activity, suggesting MDF might be partially inactivated by purification. As purified, MDF was approximately 1,000-fold less potent at deactivating macrophages than TGF-beta. Antibodies that neutralized the macrophage-deactivating effect of TGF-beta did not inhibit deactivation by MDF.     

Bound amino acids in uremic sera: search for peptidic middle molecules by preparative polyacrylamide gel electrophoresis and high performance liquid chromatography

The samples of normal and uremic sera were ultrafiltered, separated on SEP-PAK C18 cartridges and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The serum material extracted from the slab gel was purified from SDS and further fractionated by reverse-phase high performance liquid chromatography (HPLC). The obtained fractions were examined by amino acid analysis and mass spectrometry. An increased number of moderately polar fractions containing bound amino acids was found in sera of uremic patients on hemodialysis. Two most prominent uremic fractions corresponded to N-benzoylglycine (hippuric acid) (fraction I, k' = 9,4) and phenylacetylglutamine (fraction II, k' = 9,7). Increased amounts of bound glutamine, glycine, serine, leucine, asparagine, alanine, valine, phenylalanine were found in other moderately polar uremic fractions. These fractions (k' range from 12.1 to 13.2) contained no free amino acids, nor any other known small uremic serum compounds; they were considered as peptide 'middle molecules' (MM) of a molecular mass smaller than 1700.