Purification of twelve cyanogen bromide fragments from bovine plasma fibronectin and the amino acid sequence of eight of them. Overlap evidence aligning two plasmic fragments, internal homology in gelatin-binding region and phosphorylation site near C terminus

Twelve cyanogen bromide fragments (CB1-12) from bovine plasma fibronectin have been isolated and eight of these completely sequenced. Altogether they account for 502 of the total expected 1880 residues in each of the two chains of fibronectin. Four of these fragments (CB1-4) constitute residues 1-289 in fibronectin with CB4 overlapping the N-terminal 29-kDa plasmic fragment to the second plasmic fragment, of 170-kDa in fibronectin. Fragments CB 5-9 are all contained within a 45-kDa gelatin-binding region, which is N-terminal in the 170-kDa fragment. The sequence of two of these five fragments in the 45-kDa fragment (CB7-8) contains two mutually homologous stretches with 57% sequence identity. Another two fragments (CB10-11) are derived from the heparin-binding region of the 170-kDa fragment. CB12 constitutes the C-terminal 13-residue stretch in fibronectin and contains a partly phosphorylated serine residue in the C-terminal sequence: -Arg-Glu-Asp-Ser(P)-Arg-Glu.     

Human and Xenopus cingulin share a modular organization of the coiled-coil rod domain: predictions for intra- and intermolecular assembly

The complete nucleotide and derived amino acid sequences of Homo sapiens cingulin cDNA (5143 bp) were determined by sequencing two distinct EST clones that showed significant sequence homology to Xenopus laevis cingulin. Protein sequence analysis indicates that the molecule contains two chains and has a tripartite structure with N-terminal (head) domains, a coiled-coil rod domain (length, 120 nm), and short C-terminal (tail) domains. Human and Xenopus cingulin heads are only 33% identical, yet a human cingulin N-terminal fragment still interacts with canine ZO-1 and ZO-2 in vitro. The rod domain contains two A and two B subdomains, though it lacks the third B subdomain present in Xenopus cingulin. The heptad substructures of Xenopus and human cingulins were further characterized by computer analysis and indicated that the two-stranded coiled-coil structure contained chains that were parallel and in axial register. Fast Fourier transform analysis and a scoring technique designed to recognize potential interactions between different supramolecular arrangements suggests that cingulin dimers may further assemble through antiparallel interactions between the last approximately 100 amino acids of the coiled-coil region. Cingulin mRNA ( approximately 5.2 kb) was detected by Northern blotting in epithelial tissues. A human cingulin EST was mapped to chromosome 1q21 using the UniGene database.     

Intrachain disulphide bridges in immunoglobulin G heavy chains. The Fc fragment

The disulphide bridges of the Fc fragment (C-terminal half of the heavy chain) have been studied in several human immunoglobulins, containing heavy chains of different antigenic types (gamma1, gamma2, gamma3 and gamma4), and in heavy-chain-disease proteins. Two intrachain disulphide bridges were found to be present. The sequences appear to be identical in the Fc fragments of two types of chain studied (gamma1 and gamma3), and very similar to corresponding sequences of the Fc fragment in rabbit. These results suggest that the C-terminal half of the heavy chains is covalently folded (in a similar fashion to the light chains) with a C-terminal loop and an N-terminal loop. The similarity is emphasized by comparison of the sequence and location of the disulphide-bridged peptides of the C-terminal loop of heavy and light chains. The N-terminal loop, on the other hand, appears to be very different in Fc fragments and light chains. The C-terminal loop is the only one present in the F'c fragment.     

Domain analysis of cortexillin I: actin-bundling, PIP(2)-binding and the rescue of cytokinesis

Cortexillins are actin-bundling proteins that form a parallel two-stranded coiled-coil rod. Actin-binding domains of the alpha-actinin/spectrin type are located N-terminal to the rod and unique sequence elements are found in the C-terminal region. Domain analysis in vitro revealed that the N-terminal domains are not responsible for the strong actin-filament bundling activity of cortexillin I. The strongest activity resides in the C-terminal region. Phosphatidylinositol 4,5-bisphosphate (PIP(2)) suppresses this bundling activity by binding to a C-terminal nonapeptide sequence. These data define a new PIP(2)-regulated actin-bundling site. In vivo the PIP(2)-binding motif enhances localization of a C-terminal cortexillin I fragment to the cell cortex and improves the rescue of cytokinesis. This motif is not required, however, for translocation to the cleavage furrow. A model is presented proposing that cortexillin translocation is based on a mitotic cycle of polar actin polymerization and midzone depolymerization.     

Unusual proteolysis of the protoxin and toxin from Bacillus thuringiensis. Structural implications

Trypsin is shown to generate an insecticidal toxin from the 130-kDa protoxin of Bacillus thuringiensis subsp. kurstaki HD-73 by an unusual proteolytic process. Seven specific cleavages are shown to occur in an ordered sequence starting at the C-terminus of the protoxin and proceeding toward the N-terminal region. At each step, C-terminal fragments of approximately 10 kDa are produced and rapidly proteolyzed to small peptides. The sequential proteolysis ends with a 67-kDa toxin which is resistant to further proteolysis. However, the toxin could be specifically split into two fragments by proteinases as it unfolded under denaturing conditions. Papain cleaved the toxin at glycine 327 to give a 34.5-kDa N-terminal fragment and a 32.3-kDa C-terminal fragment. Similar fragments could be generated by elastase and trypsin. The N-terminal fragment corresponds to the conserved N-terminal domain predicted from the gene-deduced sequence analysis of toxins from various subspecies of B. thuringiensis, and the C-terminal fragment is the predicted hypervariable sequence domain. A double-peaked transition was observed for the toxin by differential scanning calorimetry, consistent with two or more independent folding domains. It is concluded that the N- and C-terminal regions of the protoxin are two multidomain regions which give unique structural and biological properties to the molecule.     

Localisation of light chain and actin binding sites on myosin

A gel overlay technique has been used to identify a region of the myosin S-1 heavy chain that binds myosin light chains (regulatory and essential) and actin. The 125I-labelled myosin light chains and actin bound to intact vertebrate skeletal or smooth muscle myosin, S-1 prepared from these myosins and the C-terminal tryptic fragments from them (i.e. the 20-kDa or 24-kDa fragments of skeletal muscle myosin chymotryptic or Mg2+/papain S-1 respectively). MgATP abolished actin binding to myosin and to S-1 but had no effect on binding to the C-terminal tryptic fragments of S-1. The light chains and actin appeared to bind to specific and distinct regions on the S-1 heavy chain, as there was no marked competition in gel overlay experiments in the presence of 50-100 molar excess of unlabelled competing protein. The skeletal muscle C-terminal 24-kDa fragment was isolated from a tryptic digest of Mg2+/papain S-1 by CM-cellulose chromatography, in the presence of 8 M urea. This fragment was characterised by retention of the specific label (1,5-I-AEDANS) on the SH1 thiol residue, by its amino acid composition, and by N-terminal and C-terminal sequence analyses. Electron microscopical examination of this S-1 C-terminal fragment revealed that: it had a strong tendency to form aggregates with itself, appearing as small 'segment-like' structures that formed larger aggregates, and it bound actin, apparently bundling and severing actin filaments. Further digestion of this 24-kDa fragment with Staphylococcus aureus V-8 protease produced a 10-12-kDa peptide, which retained the ability to bind light chains and actin in gel overlay experiments. This 10-12-kDa peptide was derived from the region between the SH1 thiol residue and the C-terminus of S-1. It was further shown that the C-terminal portion, but not the N-terminal portion, of the DTNB regulatory light chain bound this heavy chain region. Although at present nothing can be said about the three-dimensional arrangement of the binding sites for the two kinds of light chain (regulatory and essential) and actin in S-1, it appears that these sites are all located within a length of the S-1 heavy chain of about 100 amino acid residues.     

Iron-binding fragments from the carboxyl-terminal region of hen ovotransferrin.

1. When iron-saturated hen ovotransferrin was treated with subtilisin the N-terminal half was digested at a faster rate than the C-terminal half, allowing the latter to be isolated as a single-chain fragment of mol.wt 35000. 2. In mildly acid conditions iron-ovotransferrin loses iron preferentially from its N-terminal binding site. Trypsin digestion of the resulting monoferric ovotransferrin also gave rise to a C-terminal fragment. 3. Comparison of the N-terminal fragment with the C-terminal fragments shows differences in composition, peptide 'maps', CNBr-cleavage patterns and antigenic structures. The C-terminal fragments carry the carbohydrate group of ovotransferrin. 4. Both N-terminal and C-terminal fragments donate their bound iron to rabbit reticulocytes.     

Sequence comparisons of intermediate filament chains: evidence of a unique functional/structural role for coiled-coil segment 1A and linker L1

A comprehensive analysis of the sequences of all types of intermediate filament chains has been undertaken with a particular emphasis on those of segment 1A and linker L1. This has been done to assess whether structural characteristics can be recognized in the sequences that would be consistent with the role of each region in the recently proposed "swinging head" hypothesis. The analyses show that linker L1 is the most flexible rod domain region, that it is the most elongated structure (on a per residue basis), and that it is the most variable region as regards sequence and length. Segment 1A has one of the two most highly conserved regions of sequence in the rod domain (the other being at the end of segment 2B), with seven particular residues conserved across all chain types. It also contains one of the very few potential interchain ionic interactions that could be conserved across all chain types. However, the aggregation of chains in segment 1A is specified less precisely overall by interchain ionic interactions than are the other coiled-coil segments. The apolar residue contents in positions a and d of the heptad substructure are the highest of any coiled-coil segment in the intermediate filament family. Segment 1A also displays an amino acid composition atypical of not only coiled-coil segments 1B and 2B, but indeed of two-stranded coiled coils in general. Nonetheless, molecular modeling based on the crystal structure of the monomeric 1A fragment from human vimentin shows that coiled-coil formation is plausible. The most extensive regions of apolar/aromatic residues lie at the C-terminal end of segment 2B in the helix termination motif and in segment 1A in and close to the helix initiation motif. The predicted stability of the individual alpha-helices in segment 1A is greater than in those comprising segments 1B and 2B, though potential intrachain ionic interactions are either lacking or are minimal in number. Analysis of the 1A sequence and those regions immediately N- and C-terminal to it has shown that the capping residues are near optimal close to the previously predicted ends, thus adding to the likely stability of the alpha-helical structure. However, a second terminating sequence is predicted in 1A (about 10 residues back from the C-terminus). This allows the possibility of some unwinding of the alpha-helical structure of 1A immediately adjacent to linker L1 when the head domains no longer stabilize the coiled-coil structure. All of these data are consistent with the concept of a flexible hinge at L1 and with the ability of the two alpha-helical coiled-coil strands to separate under appropriate conditions and partly unwind at their C-terminal ends to allow the head domains a greater degree of mobility, thus facilitating function.     

The binding of lanthanides to non-immune rabbit immunoglobulin G and its fragments.

The binding of Gd(III) to rabbit IgG (immunoglobulin G) and the Fab (N-terminal half of heavy and light chain), (Bab')2 (N-terminal half of heavy and light chains joined by inter-chain disulphide bond), Fc (C-terminal half of heavy-chain dimer)and pFc' (C-terminal quarter of heavy-chain dimer) fragments was demonstrated by measurements of the enhancement of the solvent-water proton relaxation rates in the appropriate Gd(III) solutions. At pH 5.5 there are six specific Gd(III)-binding sites on the IgG. These six sites can be divided into two classes; two very 'tight' sites on the Fc fragment (Kd approx. 5 muM) and two weaker sites on each Fab region (Kd approx. 140 muM). Ca(II) does not apparently compete for these metal-binding sites. The metal-binding parameters for IgG can be explained as the sum of the metal binding to the isolated Fab and Fc fragments, suggesting that there is no apparent interaction between the Fab and Fc regions in the IgG molecule. The binding of Gd(III) to Fab and Fc fragments was also monitored by measuring changes in the electron-spin-resonance spectrum of Gd(III) in the presence of each fragment and also by monitoring the effects of Gd(III) on the protein fluorescence at 340 nm (excitation 295 nm). The fluorescence of Tb(III) solutions of 545 nm (excitation 295 nm) is enhanced slightly on addition of Fab or Fc.     

Secondary structure of component 8c-1 of alpha-keratin. An analysis of the amino acid sequence.

The amino acid sequence of component 8c-1 from alpha-keratin was analysed by using secondary-structure prediction techniques, homology search methods, fast Fourier-transform techniques to detect regularities in the linear disposition of amino acids, interaction counts to assess possible modes of chain aggregation and assessment of hydrophilicity distribution. The analyses show the following. The molecule has two lengths of coiled-coil structure, each about 20 nm long, one from residues 56-202 with a discontinuity from about residue 91 to residue 101, and the other from residues 219-366 with discontinuities from about residue 238 to residue 245 and at about residue 306. The acidic and basic residues in the coiled-coil segment between residues 102 and 202 show a 9,4-residue structural period in their linear disposition, whereas between residues 246 and 366 a period of 9.9 residues is observed in the positioning of ionic residues. Acidic and basic residues are out of phase by 180 degrees. Similar repeats occur in corresponding regions of other intermediate-filament proteins. The overall mean values for the repeats are 9.55 residues in the N-terminal region and 9.85 residues in the C-terminal region. The regions at each end of the protein chain (residues 1-55 and 367-412) are not alpha-helical and contain many potential beta-bends. The regions specified in have a significant degree of homology mainly due to a semi-regular disposition of proline and half-cystine residues on a three-residue grid; this is especially apparent in the C-terminal segment, in which short (Pro-Cys-Xaa)n regions occur. The coiled-coil segments of component 8c-1 bear a striking similarity to corresponding segments of other intermediate-filament proteins as regards sequence homology, structural periodicity of ionic residues and secondary/tertiary-structure predictions. The assessments of the probabilities that these homologies occurred by chance indicate that there are two populations of keratin filament proteins. The non-coiled-coil regions at each end of the chain are less hydrophilic than the coiled-coil regions. Ionic interactions between the heptad regions of components 8c-1 and 7c from the microfibrils of alpha-keratin are optimized when a coiled-coil structure is formed with the heptad regions of the constituent chains both parallel and in register.     

Structural study of long arm fragments of laminin. Evidence for repetitive C-terminal sequences in the A-chain, not present in the B-chains

The outer segments of the long arm of laminin have recently been shown to mediate attachment of many cell types and to stimulate neurite outgrowth. For a structural characterization of this part of the molecule we prepared, by limited elastase digestion of laminin, fragments E3 and E8, previously identified as a globular heparin-binding domain and as a 35-nm-long rod with a terminal globule, respectively. Fragment E3 is a domain adjacent to fragment E8. Both structures together comprise the complete terminal half of the long arm. Our data confirm current models, which predict that the C-terminal segments from all three chains contribute to its structure. The B chains terminate at the end of the rod like domain, while the large terminal globule is formed by A-chain structures only. In addition to fragment E3, two new fragments T1 and T2 obtained by tryptic cleavage of fragment E8 were characterized as substructures of the globular domain. Screening of a mouse cDNA library with synthetic oligonucleotides allowed isolation of an 1.8-kb cDNA clone encoding 547 C-terminal amino acids of the A chain and some 196 nucleotides of the 3'-untranslated region including a single polyadenylation site. The clone contained portions of domain T2 and the complete heparin binding domain E3 which was thus identified as the most C-terminal domain of the A chain. Sequence alignment indicated that the terminal globule is formed by homologous repeats of some 140 residues having no counterpart in the B chains.     

Isolation of a Raw Starch-Binding Fragment from Barley α-Amylase

Barley α-amylase was purified by ammonium sulfate fraction, ion-exchange, ultrafiltration, and gel filtration to homogeneity. The purified enzyme was partially digested with trypsin, and the reaction mixture was applied to a cyclohepta-amylose epoxy Sepharose 6B column. Bound fragments were eluted by free cyclohepta-amylose, lyophilized, and separated on Tricine gels. Four fragments were shown to interact with β-cyclodextrin. The fragment that could be identified on the gel with the lowest molecular weight (11 kDa) was electroblotted onto PVDF membrane for sequencing. The N-terminal sequence of this fragment was determined with the N-terminal amino acid corresponding to Ala283 in the whole protein. The trypsin cleavage was at Lys282/Ala283 and the C-terminal cleavage occurred at Lys354/Ile355 to give a fragment size of 11 kDa as estimated by SDS-PAGE. The fragment would be located at the C-terminal region, forming a majority of the antiparallel β-sheets in domain C and the α₇-and α₈-helices of the (α/β)₈ domain.     

Light chain-dependent self-association of dynein intermediate chain

Dynein light chains are bivalent dimers that bind two copies of dynein intermediate chain IC to form a cargo attachment subcomplex. The interaction of light chain LC8 with the natively disordered N-terminal domain of IC induces helix formation at distant IC sites in or near a region predicted to form a coiled-coil. This fostered the hypothesis that LC8 binding promotes IC self-association to form a coiled-coil or other interchain helical structure. However, recent studies show that the predicted coiled-coil sequence partially overlaps the light chain LC7 recognition sequence on IC, raising questions about the apparently contradictory effects of LC8 and LC7. Here, we use NMR and fluorescence quenching to localize IC self-association to residues within the predicted coiled-coil that also correspond to helix 1 of the LC7 recognition sequence. LC8 binding promotes IC self-association of helix 1 from each of two IC chains, whereas LC7 binding reverses self-association by incorporating the same residues into two symmetrical, but distant, helices of the LC7-IC complex. Isothermal titration experiments confirm the distinction of LC8 enhancement of IC self-association and LC7 binding effects. When all three light chains are bound, IC self-association is shifted to another region. Such flexibility in association modes may function in maintaining a stable and versatile light chain-intermediate chain assembly under changing cellular conditions.     

Folding properties of functional domains of tropomodulin.

Tropomodulin (Tmod) stabilizes the actin-tropomyosin filament by capping the slow-growing end (P-end). The N- and C-terminal halves play distinct roles; the N-terminal half interacts with the N-terminal region of tropomyosin, whereas the C-terminal half interacts with actin. Our previous study (A. Kostyukova, K. Maeda, E. Yamauchi, I. Krieger, and Y. Maéda Y., 2000, Eur. J. Biochem. 267:6470-6475) suggested that the two halves are also structurally distinct from each other. We have now studied the folding properties of the two halves, by circular dichroism spectroscopy and by differential scanning calorimetry of the expressed chicken E-type tropomodulin and its large fragments. The results showed that the C-terminal half represents a single, independently folded unit that melts cooperatively through a two-state transition. In contrast, the N-terminal half lacks a definite tertiary structure in solution. The binding of N11, a fragment that corresponds to the first 91 amino acids of the tropomodulin, to tropomyosin substantially stabilized the tropomyosin. This may indicate that the flexible structure of the N-terminal half of tropomodulin in solution is required for binding to tropomyosin and that the N-terminal half acquires its tertiary structure upon binding to tropomyosin.     

The structure of the major protein of the human erythrocyte membrane. Characterization of the intact protein and major fragments.

Polypeptide 3, the major membrane-penetrating protein of the human erythrocyte membrane, was characterized, together with two major fragments derived by specific proteolysis of the native protein in the membrane. One fragment (fragment 3f) was obtained from thermolysin cleavage in the extracellular region of the protein, and the other (fragment T1) was derived from tryptic cleavage in the intracellular region of the protein. The results of N- and C-terminal group analysis suggest that fragment 3f contains the N-terminal region of polypeptide 3 and fragment T1 contains the C-terminal part of the molecule. The carbohydrate contents of the polypeptides suggest that carbohydrates are present in three regions of the molecule, much of this carbohydrate being present in the C-terminal part of the molecule. This region of the protein also contains the receptors for concanavalin and the lectins from Phaseolus vulgaris and Ricinis communis, and our results suggest that there is heterogeneity in the carbohydrate chains present in the C-terminal region of polypeptide 3. These data are related to the folding of polypeptide 3 in the erythrocyte membrane.     

Characterization of domain borders and of a naturally occurring major fragment of staphylococcal alpha-toxin

A naturally occurring staphylococcal alpha-toxin fragment with an apparent membrane-binding capacity but without toxic activities is shown to be derived from the C-terminal half of the intact polypeptide chain by cleavage between position 134 and 135 in the parent molecule. The resulting N-terminus is slightly ragged with a fragment start not only at position 135 but also at the adjacent position 136. Another naturally occurring fragment starts at position 9, derived from an original cleavage between position 8 and 9 in the parent molecule. Analysis of non-purified fragment mixtures confirmed these positions and established that only one further region, at positions 71-72, is partly sensitive to proteolysis under natural conditions. Trypsin treatment has limited effects on the native toxin molecule, giving essentially only two initial cleavages with resultant large fragments. One of these cleavages is at the peptide bond between position 131 and 132, thus only three residues away from the position of the major naturally occurring cleavage. The other bond sensitive to trypsin is between position 8 and 9, thus identically positioned to the cleavage occurring naturally. Together, all the cleavages define a region in a central segment of the polypeptide chain that has all the properties of an inter-domain segment. The C-terminal half appears to constitute a membrane-binding domain, and the N-terminal half a structure needed for full biological activity, functionally subdividing the parent polypeptide chain.     

The C-terminal fragment of the precursor tail lysozyme of bacteriophage T4 stays as a structural component of the baseplate after cleavage

Tail-associated lysozyme of bacteriophage T4 (tail lysozyme), the product of gene 5 (gp 5), is an essential structural component of the hub of the phage baseplate. It is synthesized as a 63-kDa precursor, which later cleaves to form mature gp 5 with a molecular weight of 43,000. To elucidate the role of the C-terminal region of the precursor protein, gene 5 was cloned and overexpressed and the product was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, analytical ultracentrifugation, and circular dichroism. It was shown that the precursor protein tends to be cleaved into two fragments during expression and that the cleavage site is close to or perhaps identical to the cleavage site in the infected cell. The two fragments, however, remained associated. The lysozyme activity of the precursor or the nicked protein is about 10% of that of mature gp 5. Both the N-terminal mature tail lysozyme and the C-terminal fragment were then isolated and characterized by far-UV circular dichroism and analytical ultracentrifugation. The latter remained trimeric after dissociation from the N-terminal fragment and is rich in beta-structure as predicted by an empirical method. To trace the fate of the C-terminal fragment, antiserum was raised against a synthesized peptide of the last 12 C-terminal residues. Surprisingly, the C-terminal fragment was found in the tail and the phage particle by immunoblotting. The significance of this finding is discussed in relation to the molecular assembly and infection process.     

A possible difference in the heterogeneity of the heavy and light chains from a rabbit anti-p-azobenzoate antibody preparation.

CNBr cleavage of rabbit heavy (H) chains leads to the formation of a fragment, C-1, which consists of the N-terminal half of the H chain. Fragment C-1 is cleaved at methionyl residues but held together by intrachain S-S bonds so that smaller fragments can be liberated by total reduction and alkylation. In the case of the C-1 fragment from an anti-p-azobenzoate antibody preparation, which has a light (L) chain of markedly restricted heterogeneity, total reduction and alkylation liberated seven major fragments in good yield. The N-terminus of two of these fragments corresponds to position 35 of the H chain but their N-terminal sequences are clearly different. The H chain regions represented by the other fragments implied that they were derived from H chains having different distributions of methionyl residues. This hypothesis was supported by isolating six different antibody components from the antibody preparation by isoelectric focusing and then digesting them with CNBr. Comparison of the products showed that the six components all appeared to behave differently. These results are interpreted as suggesting that the process whereby H and L chains are paired in vivo may not be completely specific and may provide a simple means of generating a significant contribution to antibody diversity.     

Proteolytic hydrolysis and purification of the LRP/alfa-2-macroglobulin receptor domain from alpha-macroglobulins

A new, easier and efficient purification method, using Sephacryl and DEAE-Sephacel, of the C-terminal fragment of two alpha-macroglobulins, alpha(2)-M and PZP, is presented. Two larger peptides were identified for each protein as the C-terminal fragment, with molecular weights of approximately 30 kDa and the N-terminal sequences were determined to be SSTQDTV for alpha(2)-M and VALHLS for PZP. The smaller peptides with molecular weights of 18 kDa correspond to a shorter C-terminal sequence of these proteins, and they were determined to be EEFPFA for alpha(2)-M and ALKVQTV for PZP, with no interfering sequences detected. The results confirmed the discriminatory capacity of the purification procedure and the purity of the fragments. This new methodology facilitates biological studies of alpha-macroglobulins, and will enable elucidation of the role the C-terminal region may exert to eliminate alpha-macroglobulin-proteinases complexes from the circulation by the LRP/receptor.     

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.