Limited pepsin digestion of bovine plasma albumin

Limited pepsin digestion of bovine plasma albumin at pH 3.7 and 25 °C in the presence of octanoic acid gave two fragments, A and B, each in about 16% yield. In the absence of octanoic acid fragment A was rapidly degraded further into smaller fragments. Sodium dodecylsulfate gel electrophoreses and amino acid analyses of fragments A and B indicated their molecular weights to be about 29,000 and 34,000, respectively. Comparative studies of the cyanogen bromide peptides of fragments A and B with those of intact albumin established that fragments A and B represent, respectively, the carboxyl and the amino terminal portions of the albumin molecule.In Tris-HCl buffer (pH 7.95) at 25 °C, fragment A has one primary binding site for octanoic acid with a binding constant about one-eighth of that of albumin. This binding constant is doubled in the presence of an equimolar amount of fragment B, although fragment B itself shows very weak activity, less than one three-hundreth of that of albumin. l- and d-tryptophans competitively bind at the same primary octanoate binding site of fragment A, just as is the case with albumin.These findings together with those of other studies suggest that the albumin molecule might consist of several compact regions and that the interactions of these regions within the molecule vary with the solvent environment and upon binding of organic ligands.     

Cleavage of bacterial flagellin with cyanogen bromide. Chemical and physical properties of the protein fragments

1. Flagellin, isolated from the flagella of Salmonella adelaide, was shown by various criteria to be a pure protein. It had a molecular weight of about 40000 and contained three methionine, six tyrosine, 11 arginine and 25 lysine residues/mol., of which 11 of the lysine residues were present as in-N-methyl-lysine. 2. After treatment of flagellin with cyanogen bromide in formic acid, four main fragments (A, B, C and D) were obtained, with as many as six minor components that represented partial degradation products. The major fragments were estimated by amino acid analysis to have molecular weights of about 18000 for fragment A, 12000 for fragment B, 5500 for fragment C and 4500 for fragment D. Fragments A, B and D, but not fragment C, were recovered pure by gel chromatography as monitored by polyacrylamide-gel electrophoresis. 3. A complex between fragments C and D was also isolated (mol.wt. 10000) after limited oxidation of flagellin by chloramine-t before digestion by cyanogen bromide. After oxidation essentially only two fragments were released from flagellin by cyanogen bromide: the ;C,D' complex and a presumed ;AB' fragment. 4. The sum of the amino acid analyses of fragments A and B and the ;C,D' complex gave residue values that agreed well with the amino acid composition of native flagellin. 5. Fragments A and D contained tyrosine, and ten of the 11 in-N-methyl-lysine residues of the molecule were in fragment A. Reaction with [(125)I]iodide at small extents of substitution showed that, in flagellin, the tyrosine residue of fragment D was more readily substituted than those of fragment A. By contrast, in polymerized flagellin, the tyrosine residues of fragment A were more readily substituted. 6. Treatment of flagellin with carboxypeptidases A and B revealed the C-terminal sequence -Leu-Leu-Leu-Arg. Arginine and leucine were released by carboxypeptidase from the ;C,D' complex but not from fragment D, indicating that fragment C was C-terminal. 7. On the basis of the results from amino acid analysis, carboxypeptidase digestion, N-terminal analysis, iodination studies and polyacrylamide-gel electrophoresis, the sequence of fragments in flagellin was considered to be B-A-D-C; in the polymer, fragment A was exposed. It is suggested that methylation of the lysine residues occurred in the organism after flagellin had polymerized.     

Limited pepsin digestion of human plasma albumin.

Limited pepsin digestion of human plasma albumin at pH 3.5 and 0 degrees in the presence of octanoate caused cleavage at residue 307 of the albumin molecule to yield two fragments. Thw two fragments corresponding to the NH2- and the COOH-terminal halves of the molecule were isolated in yields of about 15%. The COOH-terminal fragment is a mixture in which about 85% of the molecules had an additional cleavage at residue 422 of the albumin molecule. The COOH-terminal fragment with the additional cleavage at residue 422 contains two peptides which are linked by a disulfide bridge at residues 391 and 437 of the albumin molecule. Both the NH2- and the COOH-terminal fragment of human albumin showed no detectable binding of octanoate anions, that is, less than 1/170 of the binding constant of the primary site of human albumin. These findings differ from earlier observations on limited pepsin digestion of bovine plasma albumin where the corresponding COOH-terminal fragment had the octanoate-binding activity, about 1/8 of the primary binding constant of bovine albumin, while the NH2-terminal fragment did not. The COOH-terminal fragment of bovine albumin did not have cleavage at residue 422 as in the corresponding fragment of human albumin. However, it is not clear that the loss of octanoate-binding activity of fragment C of human albumin is a direct consequence of the cleavage at residue 422.     

Purification and characterization of peptic fragments derived from the carboxyl-terminal half of human albumin

Utilizing a combination of conventional and affinity-chromatographic procedures, we have purified four fragments of human albumin that were generated by controlled limited proteolysis with pepsin [0.3 mM albumin; 37°C; 10 min; pH 3.51; 4.2 mM octanoate; pepsin/albumin, 1:1000 (w/w)]. These fragments have a molecular weight range of 9200-17,000 Da. Amino acid compositions, N- and C-terminal sequences, molecular weights, and other internal markers were used to determine the location of these fragments within the parent molecule. All of the fragments were shown to be derived from the C-terminal half of human albumin. The presence of multiple pepsin-sensitive bonds near the C terminus of each fragment complicated the assignment of specific residue numbers to each fragment. Two pairs of similar peptides were identified: (A) those corresponding to a single-loop structure (residues 309–380 and 309–387) and (B) those containing multiple loops and intraloop cleavages [residues 309–(491–495) with 408–423 deleted]. Purification of these fragments without disulfide bond reduction confirms portions of the loop structure of human albumin and demonstrates increased susceptibility of two specific regions of the C-terminal half of the molecule to peptic digestion.     

Partial non-cleavage by cyanogen bromide of a methionine--cystine bond from human serum albumin and bovine alpha-lactalbumin.

When human albumin was treated with CNBr, a fragment designated D was obtained and attributed to the absence from some of the albumin molecules of methionine at position 123 [Lapresle & Doyen (1975) Biochem. J. 151, 637-643]. The present study shows that methionine-123 is converted into homoserine without cleavage of the subsequent methionine-cystine bond. With bovine alpha-lactalbumin, a further example of non-cleavage of a methionine-cystine bond with conversion of methionine into homoserine is reported.     

Optical detection of triplet-state magnetic resonance studies on individual tryptophan residues of serum albumin: correlation between phosphorescence and zero-field splittings

Cyanogen bromide cleavage of bovine serum albumin (BSA) yields two fragments, N (1-183) and C (184-582), containing 183 and 399 amino acid residues, respectively. Each in each fragment are characterized in this study by phosphorescence and optically detected magnetic resonance spectroscopy, and the results are compared with those of the intact albumin. Trp-134 in fragment N is located in a hydrophobic environment in the interior of the protein, as reflected by its red-shifted phosphorescence and characteristic zero-field splittings. The spectral properties of Trp-212 in fragment C suggest its location in a partially buried, inhomogeneous environment. They show great similarity to those of human serum albumin, which contains a single Trp at position 214. The Trp phosphorescence 0,0-bands of fragments C and N are fitted with Gaussian functions by computer, and their relative contributions to the phosphoresence 0,0-band of BSA are adjusted to fit the observed BSA 0,0-band. The wavelength dependence of the [D[-[E[ transition frequencies of fragments N and C is then weighted by their 0,0-band intensity, taking into account differences in spin alignment, and summed to predict the peak frequency of the [D[-[E[ band profile as a function of phosphorescence wavelength for the intact BSA. Good agreement between predicted and observed behavior of [D[-[E[ vs. wavelength for the intact protein provides strong evidence for the additivity of the phosphorescence and ODMR spectra of the individual Trp sites in BSA. We find that Trp-134 and Trp-212 have wavelength-independent and wavelength-dependent zero-field splittings, respectively.     

Amino acid sequences of fragments I and II obtained by cyanogen bromide cleavage of rat serum albumin

The amino acid sequences of fragment I, the N-terminal cyanogen bromide fragment, and fragment II, a fragment between the first and second methionine residues, of rat serum albumin were determined by conventional methods in consideration of the sequences of human and bovine serum albumin. These sequences were compared with those of human and bovine serum albumin.     

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.     

Chemical structure of two fragments of human serum albumin and their location in the albumin molecule.

1. 'Inhibitor fragment' isolated from human serum albumin degraded by rabbit cathepsin D is composed of one peptide chain with two intrachain disulphide bonds. There are two kinds of inhibitor molecules having different N-terminal amino acids: one is threonine and the other glutamine. 2. Fragment F1, isolated from inhibitor degraded by trypsin, is composed of two chains linked by a disulphide bond. There are three kinds of fragment F1. All have one alpha chain in common, which has an intrachain disulphide bond. They differ by the nature of the chain, which is linked to the alpha chain by a disulphide bond. The epsilon chain is present in trace amounts. The two other chains, beta and gamma, differ by their C-terminal amino acid, which is respectively arginine and lysine. 3. Inhibitor is composed of the last 92 or 89 residues of the human albumin molecule and fragment F1 is composed of two parts of this C-terminal portion of the albumin molecule.     

Fragments of bovine serum albumin produced by limited proteolysis. Conformation and ligand binding.

Twelve fragments of bovine serum albumin, isolated following limited tryptic or peptic hydrolysis, have been studied to define secondary structure and locate ligand-binding sites. Based on circular dichroism, the conformational pattern of albumin (68% alpha helix and 18% beta structure) is substantially retained by individual fragments, indicating that secondary configuration is locally determined and is not destroyed during the cleavage process nor during fragment purification. The strong bilirubin-binding site of bovine serum albumin is present in 3 of the 12 fragments. Residues 186-238 are common to the three fragments and absent from those fragments which do not bind bilirubin; consequently the strong bilirubin-binding site is suggested to involve this region. By similar reasoning, the presence of palmitate-binding sites in some fragments and not in others indicates that the three strongest sites for the binding of palmitate are located in the carboxyl-terminal two-thirds of the molecule. The first site (KA approximately 2 X 10(7) M-1) is suggested as residues 377-503; the second site (KA approximately 8 X 10(6) M-1), residues 239-306; the third site (KA approximately 2 X 10(6) M-1), residues 307-377. Bromocresol Green, a reagent used in the assay of ablumin, was bound by fragments rougly in proportion to their size but showed particular affinity for the region of the strong bilirubin-binding site. The fluorescent probe, 8-anilino-1-naphthalensulfonate, was in general bound by large fragments, supporting the concept that this ligand is held principally in clefts between domains of the macromolecule.     

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.     

Immunoprecipitation is inappropriate for the isolation of radiochemically pure albumin from tissues

Rats were given intravenous injections of l-[1-¹⁴C]leucine. Twelve minutes after injection, testes, kidneys, livers, and hepatomas were excised rapidly from one group of animals bearing Morris hepatoma 5123tc. From a second group of rats, the blood was removed 90 min after injection. Tissue extracts and serum were divided into three portions each, and albumin was isolated from each of the three portions by one of three different methods.The three different isolation procedures were the following: (A) pretreatment of the tissue extracts and serum with bovine serum albumin and its specific antiserum and subsequent immunoprecipitation of the rat serum albumin, (B) direct immunoprecipitation followed by dissolving the precipitated rat serum albumin in acid/ethanol, precipitation with ether, and ion-exchange chromatography of the redissolved albumin on CM-cellulose, and (C) a modification of a procedure published previously including fractionation with trichloroacetic acid, ethanol, ether, and ammonium sulfate, chromatography on Sephadex G-100 and DEAE-cellulose, and preparative disc electrophoresis in polyacrylamide gel at pH 10.3 and pH 2.7.With method (A), radiochemically pure albumin can be obtained only from serum. Even though testis and kidney do not synthesize albumin, albumin preparations isolated by this procedure from these organs contain significant amounts of radioactivity. Specific radioactivities measured in albumin prepared by method (A) from the four tissues examined are 5–19 times larger than those in preparations isolated by method (C). Thus, method (A) is inappropriate for the isolation of radiochemically pure albumin from the tissues studied.Procedure (B) is sufficient to obtain radiochemically pure albumin from the serum as well as from the other tissues examined except liver. With liver, this method yields an albumin preparation containing 53% more radioactivity than does albumin isolated with method (C).Method (C) is the only procedure yielding radiochemically pure albumin from all sources, including liver. In liver and hepatoma, the properties of the radioactive impurities are very similar to the properties of albumin itself. Therefore, several purification steps and a careful analysis of the distribution of radioactivity among the albumin fractions after chromatographies and electrophoreses are necessary to separate radioactive impurities from the albumin from homogenates of these two sources.     

Structural characterization of two genetic variants of human serum albumin

In the present paper we report the structural characterization of two genetic mutants of human serum albumin: albumin Vanves, a very rare, electrophoretically fast variant of French origin, and albumin Verona, a slow-migrating variant which is the most frequently observed in Italy and which possesses the same electrophoretic mobility as albumin B. Both variants were isolated from the sera of healthy heterozygous subjects. Analysis of CNBr fragments by isoelectric focusing allowed us to localize the mutation to the COOH-terminal region of the molecule (residues 549-585) in both cases. The modified fragments were then isolated on a preparative scale by HPLC and subjected to tryptic digestion. Sequential analysis of the abnormal tryptic peptide, purified by HPLC, established the mutation responsible for albumin Vanves as 574 Lys----Asn and the molecular defect of albumin Verona as 570 Glu----Lys, both probably due to point mutations in the structural genes. The amino-acid substitutions found in albumins Verona and Vanves are consistent with the electrophoretic mobilities observed for the native proteins at pH 8.6.     

Procedure for the isolation of a large peptic fragment of human serum albumin

For a thorough investigation of the drug-binding behaviour and other physicochemical properties of human serum albumin, one needs large amounts of specific fragments of albumin. Such fragments were obtained by careful proteolysis of the native protein with pepsin at pH 3.70. The fast protein liquid chromatographic technique was used to find the optimum experimental conditions for the separation of the fragments. By means of anion-exchange chromatography, chromatofocusing and gel permeation, it was possible to obtain a large fragment with a relative molecular mass of 46 000. The fragment could be assigned to segment 1–387 and therefore consists of domains 1 and 2 of the albumin structure, A 1-g amount of albumin produced 50 mg of a fragment that was 98% homogeneous.     

Physical and binding properties of large fragments of human serum albumin.

Three large fragments of human serum albumin were produced by peptic digestion of the native protein [Geisow & Beaven (1977) Biochem. J. 161, 619-625]. Fragment P44 represents residues 1-386 and fragments P29 and P31 represent residues 49-307 and residues 308-584 respectively of the albumin molecule. The large N-terminal fragment P44 has a similar percentage of alpha-helix to stored defatted albumin, although the alpha-helix content of all the fragments is significantly less than that of freshly prepared albumin. The fragment P44 appears to account for all the binding of the hydrophobic probe 8-anilinonaphthalene-1-sulphonate to albumin. N-Acetyl-L-tryptophan binds to this fragment and displaces one of the bound molecules of 8-anilinonaphthalene-1-sulphonate. Bilirubin binds to fragments P44 and P29, and the complexes show similar circular-dichroism spectra to that of the complex between bilirubin and whole albumin. These results are in agreement with affinity-labeling work on albumin with reactive ligands where substitution occurs in the N-terminal region of the molecule. The sharp conformational transitional transition in albumin which is observed between pH4 and 3.5 was absent from the fragments. This isomerization, usually called the N-F transition, probably occurs in intact albumin as a result of the unfolding or separation of the C-terminal third of the protein from the remainder of the molecule.     

Isolation,characterization and effect of acidic pH on the unfolding-refolding mechanism of serum albumin domains

Three fragments,viz., BSA-CNBr_1–183, BSA-CNBr_184–582, and BSA-T_377-582 representing domains I, II + III and III of bovine serum albumin have been isolated and purified. The physicochemical properties have been investigated and compared with their parent albumin molecule. The values of Stokes radii (nm) and intrinsic viscosities (ml/g) have been determined to be 2.36, 3.30; 3.43, 4.36; and 2.40, 3.13 for the fragments BSA-CNBr_1-183 BSA-CNBr_184-582 and BSA-T_377-582 respectively. The acid induced unfolding-refolding transitions of intact albumin and the fragment BSA-T_377-582 have been shown to occur in two steps while the fragments BSA-CNBr_1-183 and BSA-CNBr_184-582 underwent single step transitions. The formation of the acid denatured states of intact albumin, BSA-CNBr_1–183 and BSA-CNBr_184-582 was accompanied by an increase of about 86, 56 and 44% in the values of intrinsic viscosities respectively. Since all the transitions were reversible, the values of equilibrium constants,K _D, were calculated. The analysis of the dependence ofK _D on pH indicated that the first transition (N-X) of albumin was caused due to the uptake of about 3 protons by the native albumin. The intermediate state,X, is converted to acid unfolded state,D, by taking up another two protons. A comparision of the results on intact albumin with that of its fragments revealed that the second transition of the fragment BSA-T_377–582 and the two single step transitions of the fragment BSA-CNBr_1-183 and BSA-CNBr_184-582 were much closer to the second transition (X-D) of the intact albumin. The first transition of albumin has been attributed to its domain III represented by the fragment BSA-T_377-582.     

Binding and relaxometric properties of heme complexes with cyanogen bromide fragments of human serum albumin

The spectroscopic and reactivity properties of hemin complexes formed with cyanogen bromide fragments B (residues 1-123), C (124-298), A (299-585), and D (1-298) of human serum albumin (HSA) have been investigated. The complex hemin-D exhibits binding, spectral, circular dichroism, and reactivity characteristics very similar to those of hemin-HSA, indicating that fragment D contains the entire HSA domain involved in heme binding. The characteristics of the other hemin complexes are different, and a detailed investigation of the properties of hemin-C has been carried out because this fragment contains the HSA binding region of several important drugs. Hemin-C contains a low-spin Fe(III) center, with two imidazole ligands, but the complex undergoes a reversible structural transition at basic pH leading to a high-spin, five-coordinated Fe(III) species. This change determines a marked increase in the relaxation rate of water protons. Limited proteolysis experiments and mass spectral analysis carried out on fragment C and hemin-C show that the region encompassing residues Glu-208 to Trp-214 is protected from activity of proteases in the complex and, therefore, is involved in the interaction with hemin. A structural model of fragment C enables us to propose that His-242 and His-288 are the axial ligands for the Fe(III) center.     

Amino acid sequence studies on cyanogen bromide peptides of chicken caldesmon which bind to calmodulin

Three major calmodulin-binding cyanogen bromide peptides (fragments A, B, and D) were isolated from chicken gizzard muscle caldesmon and their amino acid sequences were determined. The molecular masses of fragments A, B, and D were estimated to 16, 12, and 9 kDa, respectively, by SDS-urea polyacrylamide gel electrophoresis. Fragment A was composed of 102 amino acid residues and contained homoserine at the C terminus. The amino acid sequence from the 37th residue of fragment A corresponds to the N-terminal sequence of the 15 kDa peptide which was obtained by thrombin digestion [Mornet, D., Audemard, E., & Derancourt, J. (1988) Biochem. Biophys. Res. Commun. 154, 564-571]. Thrombin 15 kDa peptide binds to F-actin but does not bind to calmodulin. Thus the N-terminal 36 residues and the C-terminal part from the 37th residue of fragment A are supposed to bind to calmodulin and F-actin, respectively. The sequences of fragments B and D were identical, but fragment D was composed of 64 amino acid residues and ended with tryptophan, whereas fragment B was of 98 or 99 amino acid residues and ended with proline. Both fragments B and D are supposed to be the C-terminal peptides of chicken caldesmon. Fragment B had heterogeneous sequences at the C-terminal region. These results can explain the reported heterogeneity of chicken caldesmon in charge and molecular mass.     

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.