L. Morávek

Complete amino acid sequence of human serum albumin

The complete understanding of the binding and transport functions of human serum albumin, the main protein constituent of plasma, requires the knowledge of its chemical structure..The results of sequence studies on human serum albumin reported till the end of 1970 have been summarized in the Atlas of Protein Sequence and Structure [ 11; the largest continuous part of the sequence known by then was that of the 24-residue N-terminal region [2,3]. These studies had shown that the heterogeneity of albumin preparations [4,5] does not originate in its primary structure. In 1970 we began in this Laboratory systematic studies of human serum albumin aimed at the determination of the complete amino acid sequence of the protein. The primary structure of human serum albumin was concurrently studied also at the University of Buffalo [6,8], at Craighton University Medical School [9], and at the University of Texas [IO]. This paper reports on the complete amino acid sequence of human serum albumin, including the positions of all amides; the results presented are confronted with sequential data reported from other laboratories.

Complete amino acid sequence of hog pepsin

The amino acid sequence of hog pepsin or of some parts of its polypeptide chain has been studied in a number of laboratories. The most recent summary of the results obtained is presented in the 1972 issue of the Atlas of Protein Sequence and Structure [ 11. Our aim has been to determine the complete covalent structure of this enzyme and thus to provide, together with the complete structures of bovine trypsinogen [2] and chymotrypsinogen [3] elucidated in this laboratory earlier, complete and independent sequence information on the fundamental proteolytic enzymes of the digestive tract. The determination of the complete structure of the whole molecule was based on the results of sequential studies on large fragments designated CB [4] obtained by cyanogen bromide cleavage of pepsin at its four methionine residues [5] (fig. 1). The studies on the individual cyanogen bromide fragments were begun after the determination of the disultide bonds of the 6 half-cystine residues of pepsin [6]. The complete amino acid sequence of fragment CBl [7], representing the 37-residue C-terminal part of the molecule, was determined. Later, the 55residue N-terminal amino acid sequence of pepsin [8,9] was

Haeme binding to human serum albumin and to the three large cyanogen bromide albumin fragments

Abstract 1. 1. The interaction of haeme with albumin and CNBr albumin fragment N (amino acid residues 1–123), M (124–298) and C (299–585) was investigated by means of difference spectroscopy, circular dichroism and stopped now measurements. 2. 2. The single binding site for haeme was found on each of the fragments with the binding constant K a values: N fragment, 5.4 × 10 4 M −1 ; M fragment, 6.5 × 10 4 M −1 ; C fragment, 2.7 × 10 4 M −1 . 3. 3. The primary haeme binding site of albumin molecule ( K a = 5 × 10 4 M −1 , Beaven et al ., 1974) is probably located on M fragment with C fragment co-operating in formation of its structure.

The characterization of two new low molecular weight proteins (LMPs) from Streptococcus pyogenes.

Two novel extracellular mitogenic substances were isolated from Streptococcus pyogenes strain NY-5 and characterized. The purification steps involved an initial enrichment of the proteins from culture supernatant by silica gel adsorption, followed by ion exchange chromatography and gel filtration. The purified materials were homogeneous in SDS-PAGE, showed estimated molecular weights of 12 kD and isoelectric points of 4.7 and 4.3, respectively. Both proteins (LMP-12k-4.3pI and LMP-12k-4.7pI) demonstrated lymphocyte transformation activity at a concentration of 0.1 microgram/ml. The LMP-12k-4.7pI showed a 69.2% homology of the amino acid sequence with that of a phosphocarrier protein of Staphylococcus aureus and with a total identity in the active centre. The same protein was also isolated from streptococcal group C strain H46A with an N-terminal amino acid sequence being identical. The LMP-12k-4.7pI demonstrated biochemical properties identical with those of the earlier described streptococcal pyrogenic exotoxin type D. The LMP-12k-4.3pI did not show such a clear relation to other functional proteins.

Purification and characterization of streptococcus pyogenes erythrogenic toxin type a produced by a cloned gene in streptococcus sanguis

The gene of Streptococcus pyogenes erythrogenic toxin type A (speA) has been previously cloned in Streptococcus sanguis (Challis) and produces extracellular erythrogenic toxin type A (ET A). The ET A produced and secreted by this heterologous host was purified to homogeneity and shown to have properties identical to ET A produced by S. pyogenes strain NY-5; i.e., serological identity in immunodiffusion, migration in SDS-polyacrylamide gel electrophoresis, mitogenic activity, inhibition of mitogenic activity by specific antibody, and precipitation by an international scarlatina antitoxin preparation. The cloned speA gene specified an ET A which had a molecular weight identical to that of ET A from S. pyogenes previously reported from this laboratory. NH2-terminal sequence determination of the purified protein showed the first nine residues to be gln gln asp pro asp pro ser gln leu; this is consistent with predictions made from the nucleotide sequence of the speA gene according to Weeks and Ferretti and different from the sequence published by Johnson et al.

Type 1 M protein of Streptococcus pyogenes: N-terminal sequence and peptic fragments

Limited proteolysis of the surface of type 1 Streptococcus pyogenes by pepsin gives rise to fragment Pep M1 of M r 20270 as the main product which covers the N‐terminal part of the M protein. The amino acid sequence was determined of the N‐terminal region of the M protein representing the most exposed part of the molecule on the surface fibrils of streptococcal cells, which seems to be very important for the differentiation of the individual serological types. The sequence differs from the homologous N‐terminal sequences of types 5, 6 and 24, and shows a homology with sequences repeating in the chain of type 24. Fragment Pep M1 binds to fibrinogen; the absence of its 30 N‐terminal amino acid residues, however, abolishes this interaction which is believed to play a role in the virulence of S. pyogenes.

Amino acid sequence of the N-terminal region of human hemopexin

Cyanogen bromide digestion of hemopexin at its 6 methionine residues results in 7 fragments (CB1–CB7) partially connected by disulfide bridges. By sequence studies of fragments CB1‐CB4 and peptides prepared by their enzyme cleavage, a continuous amino acid sequence of the N‐terminal region of human hemopexin, comprising 220 amino acid residues, was determined. The presence of intramolecular disulfide bonds, connecting half‐cystine residues and , was proved in fragments CB2 and CB3. Fragments CB1–CB4 include 5 sites, where hexosamine oligosaccharides are attached (positions 1,41,164, 217 and probably 223). In the sequenced region two sites sensitive to acid hydrolysis ‐ bonds ⋯ Asp‐Pro ⋯ in positions and were found. In spite of the fact that pooled material of many donors was studied, no sequence heterogeneity was discovered.

Isolation of human haemopexin by bioaffinity chromatography on haeme-sepharose

A preparative procedure was developed for the isolation of human apohaemopexin from Cohn fraction IV or blood serum, based on bioaffinity chromatography on haeme-Sepharose. The isolation is carried out in the pH range 4-8; hence the possibility of degradation of the carbohydrate moiety of the glycoprotein in the acidic media used in other isolation procedures is decreased. Owing to the conditions of the separation and the good stability of the affinity support, the column can be used repeatedly for long periods without a significant loss of binding capacity. The reversibility of the conformational changes that haemopexin undergoes in acidic media was examined by hydrophobic chromatography. The original hydrophobic characteristics were restored only approximately 48 h after haemopexin had been brought into a neutral medium.

Characterization and arrangement of tryptic fragments from N-terminal region of hog pepsin.

The cyanogen bromide hydrolysis of hog pepsin at the methionine residues [1] gives rise to a large fragment which involves the N-terminal half of its chain, lacking arginine and lysine residues [2,3]. For additional specific cleavage it is convenient to aminoethylate the half-cystine residues [4, 5] as has been described for the whole pepsin molecule [6]. Trufanov et al. [7] aminoethylated the N-terminal cyanogen bromide fragment of pepsin and isolated from its tryptic digest the N-terminal peptide which they assumed to contain 105 amino acid residues. In this study the tryptic fragments derived from the aminoethylated N-terminal cyanogen bromide fragment [2] were isolated and their sequence in the chain determined. The phosphoserine residue contained in pepsin [8] was localized and material was obtained for sequence studies on the N-terminal half of the molecule of this enzyme.

Discontinuous recycling chromatography

Abstract A chromatography technique whereby the effluent is recycled in open one-column systems and which avoids the remixing of components of considerably different elution volumes is described.