The molecular defect of albumin Castel di Sangro: 536 Lys----Glu

Albumin Castel di Sangro is a rare fast-moving variant of human serum albumin which has been discovered in heterozygous form in the serum of an 85-year-old woman living in Castel di Sangro (Abruzzo, Italy). Isoelectric focusing analysis of CNBr fragments from the purified variant allowed us to localize the mutation in fragment CNBr VI (residues 447-548). This fragment was isolated on a preparative scale and subjected to tryptic digestion. Sequential analysis of the abnormal tryptic peptide, purified by reverse-phase and cation-exchange HPLC, revealed that the variant arises from the substitution of lysine 536 by glutamic acid. This amino acid replacement, probably due to a single-base substitution in the structural gene, causes a change in the net charge of -2 units, which is in keeping with both the increased electrophoretic mobility of the native protein and the isoelectric point of the modified CNBr fragment.     

The molecular defect of albumin Tagliacozzo: 313 Lys----Asn

Albumin Tagliacozzo is a fast-moving genetic variant of human serum albumin found in 19 unrelated families. The protein was isolated from the serum of a heterozygous healthy subject. Analysis of CNBr fragments by isoelectric focusing allowed us to localize the mutation to CNBr fragment IV (residues 299-329). This fragment was isolated on a preparative scale and subjected to tryptic digestion. Sequential analysis of the abnormal tryptic peptide, purified by RP-HPLC, revealed the variant was caused by 313 Lys----Asn substitution, probably due to a point mutation in the structural gene. The lack of a lysine residue accounts for the electrophoretic behavior of albumin Tagliacozzo.     

Structural characterization of a chain termination mutant of human serum albumin

Mutant forms of human serum albumin have been detected on the basis of their abnormal electrophoretic mobility which is either faster or slower than that of normal albumin. In the present work we have studied the structure of a slow variant, referred to as albumin Ge/Ct, in order to define the cause of its genetic abnormality. The protein was isolated from the serum of a young healthy woman homozygous for the variant. Analysis of CNBr fragments by isoelectric focusing allowed us to localize the mutation to the COOH-terminal region of the molecule (residues 549-585). This fragment was isolated on a preparative scale and subjected to tryptic digestion. All tryptic peptides were purified by reverse-phase high performance liquid chromatography and characterized. Sequential analysis of three abnormal peptides revealed that albumin Ge/Ct has a shortened chain with the following COOH-terminal sequence: Leu-Val-Ala-Ala-Ser-Lys580-Leu-Pro. The presence of an additional lysine residue accounts for the electrophoretic behavior of the variant. It is likely that the variant may be caused by a single base deletion in the structural gene, a Cyt in mRNA codon 580, and the consequent shift in reading frame.     

Two alloalbumins with identical electrophoretic mobility are produced by differently charged amino acid substitutions.

We describe the amino acid substitutions of albumins Sondrio and Paris 2, two slow moving variants of human serum albumin, which show an identical electrophoretic mobility on cellulose acetate at three different pH values. These variants have been found in several instances in a wide geographic area including Northern Italy and France. Both alloalbumins were isolated from the sera of heterozygous subjects. Isoelectric focusing analysis of CNBr fragments from the purified variants allowed us to localize the mutation of albumin Sondrio in fragment CNBr V (residues 330-446) and that of albumin Paris 2 in CNBr VII (residues 549-585). Sequential analysis of the variant CNBr VII established the molecular defect of albumin Paris 2 as 563 Asp----Asn. Fragments CNBr V from normal and Sondrio albumins were isolated on a preparative scale and subjected to tryptic and V8 proteinase digestion. Sequence determination of the abnormal tryptic and V8 peptides revealed that the variant arises from the substitution of glutamic acid 333 by lysine. Thus, a +1 change in the C-terminal region of the albumin molecule produces a variant with the same electrophoretic mobility as an alloalbumin with a +2 substitution in the central domain, suggesting a higher degree of exposure to the solvent of the C-terminal tailpiece. Both amino acid substitutions are consistent with a G----A transition in the first position of the corresponding codon in the structural gene.     

Genetic variants of human albumin: structural characterization of allotypes used as references for electrophoretic classification

Eight different types of genetic variants of albumin are observed in the French population. The analysis of electrophoretic patterns of sera containing these variants, performed a three different pHs (8.6, 5.0 and 6.9) after addition of a reference protein (transferrin), allows the identification each variant by a quantitative estimation of its relative mobilities. The accuracy and reproducibility of the technique make it a useful reference method, commonly employed for studying European variants. The samples used as references for five genetic variant types, proalbumins Christchurch and Lille, albumins Vanves, B and Reading, were subjected to sequence analysis to determine the nature and localization of their structural change. Together with the mutations of albumins Gent and Roma previously described, the data presented here make available seven reference specimens for which the structural changes are characterized out of the eight variants known to exist in France.     

Structural characterization, stability and fatty acid-binding properties of two French genetic variants of human serum albumin

Four bisalbuminemic, unrelated persons were found in Bretagne, France, and their variant and normal albumins were isolated by DEAE ion exchange chromatography, reduced, carboxymethylated and treated with CNBr. Comparative two-dimensional electrophoresis of the CNBr digests showed that three of the variants were modified in fragment CB4, whereas the fourth had an abnormal fragment CB1. These fragments were isolated, digested with trypsin and mapped by reverse-phase HPLC. Sequencing of altered tryptic peptides showed that the three variants modified in CB4 were caused by the same, previously unreported, amino acid substitution: Asp314-->Val (albumin Brest). The fourth, however, was a proalbumin variant with the change Arg-2-->Cys (albumin Ildut). Both amino acid substitutions can be explained by point mutations in the structural gene: GAT-->GTT (albumin Brest) and CGT-->TGT (albumin Ildut). The proalbumin Ildut is very unstable and already in vivo it is to a large extent cleaved posttranslationally to Arg-Albumin and normal albumin. Furthermore, we observed that during a lengthy isolation procedure the remaining proalbumin was changed to Arg-Albumin or proalbumin lacking Arg-6. In addition, part of normal albumin had lost Asp1. Gas chromatographic investigations using isolated proteins indicated that albumin Brest has improved in vivo fatty acid-binding properties, whereas the structural modification(s) of albumin Ildut does not affect fatty acid binding.     

Studies on a genetically determined albumin dimer

An electrophoretic variant of human albumin has been characterized as a dimer involving both disulfide and noncovalent bonds. The variant protein was isolated by starch block electrophoresis and gel filtration and extensively investigated by gel electrophoresis and immunoelectrophoresis under a variety of conditions. No significant differences were found between normal albumin and the propositus's albumin in sulfhydryl reactivity or content or in the tryptic fingerprint.Contribution No. 140 from the Blood Research Laboratory, American National Red Cross.     

Albumin Hawkes Bay; a low level variant caused by loss of a sulphydryl group at position 177

A slow migrating minor albumin component, representing 5% of total circulating albumin, was detected by routine serum protein electrophoresis and immunofixation. After treatment with 5 mM dithiothreitol the abnormal component was found to migrate normally suggesting the attachment of some component to the free thiol at position 34. However, purification and analysis by SDS-PAGE showed that the abnormal component had a slightly lower apparent molecular weight than normal albumin. Limited tryptic cleavage indicated the abnormal site to be in the N-terminal third of the molecule. HPLC analysis of tryptic peptides from this domain showed the presence of a new peptide of sequence Ala-Ala-Phe-Leu-Leu-Pro-Lys, indicating either a point mutation of 177 Cys → Phe or the deletion of residues 166–177. DNA sequencing of PCR-amplified DNA confirmed the former Cys → Phe substitution by indicating a point mutation of C to A at nucleotide position 5185. It appears that the aberrant electrophoretic mobility of the variant might be due to a gross conformational change associated with the formation of a new disulphide bond between Cys-168 and Cys-124.     

Protein and DNA sequence analysis of a ‘private’ genetic variant: albumin Ortonovo (Glu-505 → Lys)

Albumin Ortonovo is a slow moving variant of human serum albumin which has been found only in people coming from the small villages of Ortonovo and Nicola (Liguria, Italy) and reaches polymorphic frequency (≥1%) in the poorly admixed population group living in that area. This is the first report of a ‘private’ varint detected in a Caucasin population. It probably originated as a mutation in a founder individual many generations ago. Isoelectric focusing analysis of CNBr fragments from the purified variant localized the mutation in fragment CNBr (residues 447–548). This fragment was isolated on a preparative scale by reversed-phase HPLC and subjected to V8 proteinase digestion. Sequence analysis of the abnormal V8 peptide revealed that the variant arises from a previously unreported substitution at position 505 where glutamic acid has been replaced by lysine. The protein data were confirmed by DNA sequence analysis which indicated a single nucleotide change of $\text{G}\text{AA}\text{→}\text{A}\text{AA}$ in the corresponding codon of the structural gene. Since the amino acid substitution found in albumin Ortonovo accords with its electrophoretic mobility on cellulose acetate, residue 505 is probably exposed to the solvent. The clustering of the mutations in the intersubdomain connection linking subdomains IIIA and IIIB (residues 492–511) accords with the fact that this region lies on the molecular surface and is accessible to solvent.     

The molecular abnormality of albumin Parklands: 365 Asp----His

An electrophoretically slow albumin variant was isolated from the plasma of a patient with bisalbuminemia. Reverse-phase peptide mapping revealed a single altered peak when tryptic digests of the normal and variant albumin were compared. After rechromatography and amino acid analysis, a sequence/composition of Cys-Cys-Ala-Ala-Ala-His-Pro,His,Glu,Cys,Tyr,Ala,Lys was obtained for the mutant peptide, while a sequence of Cys-Cys-Ala-Ala-Ala-Asp-Pro-His-Glu-Cys-Tyr,Ala,Lys was obtained for the normal peptide. This establishes the mutation as 365 Asp----His and the new albumin has been named albumin Parklands. Interestingly, this mutation results in the loss of the single Asp-Pro bond that is normally present between residues 365 and 366. Predictably, this confers on albumin Parklands a greater resistance to partial acid hydrolysis, a feature which, when employed together with SDS-gel electrophoresis, can be used as a diagnostic test for the presence of this variant.     

Detection of a genetic variant, lysine→glutamic acid at position 372 of human serum albumin, by capillary electrophoresis and structural identification

A genetic variant of human serum albumin (alloalbumin) is detected by capillary electrophoresis (CE). Two albumin peaks, which were in the ratio of approximately one, were clearly separated. One of the peaks had the same migration time as normal albumin (Alb A) and the other (Alb X) had a longer migration time. SDS-polyacrylamide gel electrophoresis of CNBr fragments (CB) of Alb X indicated that the amino acid substitution was localized in the CB5 fragment (residue 330–446) of the molecule, because of anomalous migration of CB5 in the gel. The CE mapping of the tryptic peptides from the variant CB5 revealed clearly the existence of a new peptide, and the lack of two normal peptides. The sequence analysis of the variant peptide collected by CE micropreparation showed that the N-terminus of the variant peptide corresponded to that of T49 in Alb A. The substitution site, lysine→glutamic acid at the position 372, was revealed by sequence determination of the variant peptide purified by reversed-phase HPLC.     

Structural study of hemoglobin Hazebrouck, beta 38(C4)Thr----Pro. A new abnormal hemoglobin with instability and low oxygen affinity

A new beta-variant has been detected and structurally defined in a French male, with a life-long history of hemolytic anemia. This variant is moderately unstable and has a low oxygen affinity. The abnormal hemoglobin was not detected by standard electrophoretic procedures. It moved slightly slower than Hb A during isoelectric focusing (IEF). Two minor fractions were also seen; the first migrated just cathodal to Hb F, as did partially oxidized Hb A or hemichrome derivatives of some unstable hemoglobins; the second in the position of free alpha-chains. The abnormal beta-chain was readily separated from both beta A- and alpha A-chains by acid-urea-Triton globin chain electrophoresis. Structural study was conducted simultaneously by fingerprinting and high-performance liquid chromatography (HPLC) of tryptic peptides. A new mutation beta 38(C4)Thr----Pro was found, which was named Hb Hazebrouck.     

Human albumin variants. Nomenclature of allotypes observed in Europe and quantitative estimation of their relative mobilities

In order to clarify the actual nomenclature of the albumin allotypes in French and Italian populations we compared the samples collected in our laboratory to those kindly supplied by Dr. Porta (Italy) with reference to albumin variants classified in starch gel electrophoresis using three buffer systems by Weitkamp. The inherited human albumin variants can be classified on the basis of their relative mobilities on cellulose acetate electrophoresis compatively to human transferrin mobility. The relative mobility of each variant can be expressed by the following ratio: migration distance of the variant versus migration distance of the normal albumin where zero represents the transferrin mobility. Using three buffers system at pH 8.6, 5.0 and 6.9, it is possible to distinguish some albumin variants having a same mobility at alkaline pH and different mobilities at acidic pH. In the european area, eleven albumin variants are distinguishable on the basis of their relative mobilities at different pH: four Fast moving variants: Gent, Vanves (a new variant described here), Reading and CN/BL, and seven Slow moving variants: MI/MI Slow, GE/CT, SO/BS (or D type), Pollibauer, Gainesville, Roma and B type. Thirty-six sera from unrelated subjects with genetic bisalbuminemia were analyzed in our laboratory. Their distribution was as follows: B type (22), Pollibauer (9), SO/BS (2), Gainesville (1), Gent (1) and Vanves type (1). The frequency of bisalbuminemia was 0.35 per 1,000 in a population of 19,949 blood donors.     

Non-enzymatic posttranslational modifications of bovine serum albumin by oxo-compounds investigated by chromatographic and electrophoretic methods

Non-enzymatic posttranslational modifications of bovine serum albumin (BSA) by oxo-compounds, particularly glucose, ribose, glyoxal and glutardialdehyde, have been investigated using a set of modern chromatographic and electrophoretic separation methods. High-performance liquid chromatography (HPLC) alternatively with UV spectrophotometric (diode array) or mass spectrometric (MS) detection, polyacrylamide gel electrophoresis (PAGE) with Coomassie brilliant blue staining detection, and capillary zone electrophoresis (CZE) with UV spectrophotometric detection have been employed for the investigation of the chemical and structural changes of BSA caused by its reaction with the above oxo-compounds exhibiting different degree of reactivity. The extent of modifications was found to be dependent on the nature of the oxo-compound used and progressed in the glucose相似文献   

Enhanced Sequence Coverage in Tryptic Fragment Analysis by Two-Dimensional HPLC/MS Using a Monolithic Silica Capillary Column

The HPLC/MS system, in which a monolithic silica capillary column is directly connected to an electronspray-ionization mass spectrometer, showed superior performance at high mobile phase linear velocity. A two-dimensional (2D) HPLC/MS system was established, using an ion-exchange particle-packed capillary column at the first dimension and a monolithic silica capillary column at the second dimension. In an analysis of tryptic fragments from bovine serum albumin, an 81% sequence coverage, obtained by the 2D-HPLC/MS system, increased by 23% as compared to a 1D-HPLC/MS system. This 2D-HPLC/MS system using a monolithic silica capillary column should be useful for enhancing sequence coverage of tryptic fragments in proteomics.     

Enhanced sequence coverage in tryptic fragment analysis by two-dimensional HPLC/MS using a monolithic silica capillary column

The HPLC/MS system, in which a monolithic silica capillary column is directly connected to an electronspray-ionization mass spectrometer, showed superior performance at high mobile phase linear velocity. A two-dimensional (2D) HPLC/MS system was established, using an ion-exchange particle-packed capillary column at the first dimension and a monolithic silica capillary column at the second dimension. In an analysis of tryptic fragments from bovine serum albumin, an 81% sequence coverage, obtained by the 2D-HPLC/MS system, increased by 23% as compared to a 1D-HPLC/MS system. This 2D-HPLC/MS system using a monolithic silica capillary column should be useful for enhancing sequence coverage of tryptic fragments in proteomics.     

Structural organization of the human glucocorticoid receptor determined by one- and two-dimensional gel electrophoresis of proteolytic receptor fragments

The structural organization of the steroid-binding protein of the IM-9 cell glucocorticoid receptor was investigated by using one- and two-dimensional gel electrophoresis of proteolytic receptor fragments. One-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of receptor fragments isolated after trypsin digestion of immunopurified [3H]dexamethasone 21-mesylate ([3H]DM-) labeled receptor revealed the presence of a stable 26.5-kilodalton (kDa) steroid-containing, non-DNA-binding fragment, derived from a larger, less stable, 29-kDa fragment. The 26.5-kDa tryptic fragment appeared to be completely contained within a 41-kDa, steroid-containing, DNA-binding species isolated after chymotrypsin digestion of the intact protein. Two-dimensional electrophoretic analysis of the [3H]DM-labeled tryptic fragments resolved two (pI congruent to 5.7 and 7.0) 26.5-kDa and two (pI congruent equal to 5.7 and 6.8) 29-kDa components. This was the same number of isoforms seen in the intact protein, indicating that the charge heterogeneity of the steroid-binding protein is the result of modification within the steroid-containing, non-DNA-binding, 26.5-kDa tryptic fragment. Two-dimensional analysis of the 41-kDa [3H]DM-labeled chymotryptic species revealed a pattern of isoforms more complex than that seen either in the intact protein or in the steroid-containing tryptic fragments. These results suggest that the 41-kDa [3H]DM-labeled species resolved by one-dimensional SDS-PAGE after chymotrypsin digestion may be composed of several distinct proteolytic fragments.     

A nucleotide insertion and frameshift cause albumin Kénitra, an extended and O-glycosylated mutant of human serum albumin with two additional disulfide bridges.

Albumin Kenitra is a new type of genetic variant of human serum albumin that has been found in two members of a family of Sephardic Jews from Kenitra (Morocco). The slow-migrating variant and the normal protein were isolated by anion-exchange chromatography and, after treatment with CNBr, the digests were analyzed by two-dimensional electrophoresis in a polyacrylamide gel. The CNBr peptides of the variant were purified by reverse-phase high performance liquid chromatography and submitted to sequence analysis. Albumin Kenitra is peculiar because it has an elongated polypeptide chain, 601 residues instead of 585, and its sequence is modified beginning from residue 575. DNA structural studies showed that the variant is caused by a single-base insertion, an adenine at nucleotide position 15 970 in the genomic sequence, which leads to a frameshift with the subsequent translation to the first termination codon of exon 15. Mass spectrometric analyses revealed that the four additional cysteine residues of the variant form two new S-S bridges and showed that albumin Kenitra is partially O-glycosylated by a monosialylated HexHexNAc structure. This oligosaccharide chain has been located to Thr596 by amino-acid sequence analysis of the tryptic fragment 592-597.     

Structural differences between albumin A and albumin C of the house mouse,Mus musculus

Two albumins, albumin A from C3H mice and albumin C isolated from descendents of the wild mice in which the variant was first uncovered, were found to differ in their electrophoretic properties. Albumin C was shown to bind two more H⁺ ions than albumin A at pH 5.4. Peptide mapping after trypsin digestion revealed that albumin C had three peptides (TP-C1, TP-C2, and TP-C3) which were missing in albumin A. The latter likewise had a peptide (TP-A1) which was not found in albumin C. An amino acid analysis of the variant peptides suggests that TP-A1 had been split into TP-C1 and TP-C2 on digestion with trypsin, because a glutamic acid in TP-A1 was replaced by a lysine. This change would also appropriately alter the electrophoretic properties of albumin C. No obvious counterpart was discovered for TP-C3 of albumin C in albumin A.This work was supported by a grant from the National Research Council of Canada.     

Identification of lysine residue 199 of human serum albumin as a binding site for benzylpenicilloyl groups

Cyanogen bromide and tryptic fragments of penicilloylated serum albumin from penicillin-treated patients were separated by HPLC. Determinations of benzylpenicilloyl (BPO) were performed on the different fractions. A BPO containing peptide was identified by its amino acid sequence and the bound BPO group was located on the lysine residue 199.