Jui-Yoa Chang

The complete amino acid sequence of hirudin, a thrombin specific inhibitor: Application of colour carboxymethylation

Color carboxymethylation of cysteine residues with a new chromophoric reagent dimethylaminoazobenzene iodoacetamide, was applied to the micro‐sequence analysis of hirudin, a thrombin specific inhibitor. Six cysteine residues of the reduced hirudin were detected as colored phenylthiohydantoin derivative and 3 tryptic peptides of hirudin (all containing cysteines) were isolated as colored peptide. The complete hirudin sequence, including 6 uncertain positions left in the previous report [Petersen T.E. (1976) in: Protides of the Biological Fluids; 23rd Colloquium, pp. 145, Pergamon Press, London] was established.

The functional domain of hirudin, a thrombin-specific inhibitor

Hirudin is a thrombin‐specific of M r 8000 (65 amino acid residues). Native hirudin contains 3 disulfide linkages within the first 39 amino‐terminal residues, and highly C‐terminal segment which is freely accessible to enzyme digestion by both endo‐ and exo‐peptidases. Removal of the acidic C‐terminal amino acids of native hirudin by both chemical and enzymatic methods resulted in a concomitant loss of hirudin inhibition activity. It is concluded that this acidic C‐terminal segment of hirudin is essential for hirudin—thrombin interaction. The implication of the hirudin—thrombin interaction for the enzymatic specificity of thrombin is also discussed.

Isolation and NH2-terminal amino acid sequences of rat serum carrier proteins for insulin-like growth factors.

Three N-glycosylated carrier proteins (CP) for insulin-like growth factors (apparent molecular weights 30-32, 42 and 45 kDa) were isolated from adult rat serum. They share the same amino terminus (up to amino acid 31) and are constituents of the growth hormone-dependent native 150-200 kDa IGF carrier complex. Residues 12-31 display 60 and 50% sequence homology, respectively, to residues 2-21 of fetal rat and to residues 4-22 of a human amniotic fluid IGF carrier protein. No homology exists with the type I or II IGF receptors. Adult rat serum also contains a fourth IGF CP (24 kDa) whose 9 NH2-terminal amino acids are identical to those of the fetal form. Our findings suggest that the three N-glycosylated components originate from the same IGF carrier protein (adult form) and that the 24 kDa protein is a separate (fetal) species.

Sequence determination of a peptide fragment from electric eel acetylcholinesterase, involved in the binding of quaternary ammonium

Specific photoaffinity labelling of purified electric eel acetylcholinesterase by 3H‐labelled p‐(N,N‐dimethyl‐amino) benzenediazonium fluoroborate allows the identification of a labelled peptide fragment which is described as being involved in the binding of quaternary ammonium ions on this enzyme. Denaturation and proteolytic cleavage of the inactivated enzyme gave a mixture of peptide fragments. The purification of one labelled fragment, containing over 15% of the radioactivity incorporated in the enzyme, led to the following sequence: Gly‐Ser‐X‐Phe. The relatively low amount of this tetrapeptide did not allow us to determine the nature of the labelled residue X.

Direct analysis of the disulfide content of proteins : methods for monitoring the stability and refolding process of cystine-containing proteins

So far, the cystine (disulfide) content of proteins has been routinely determined by indirect methods, i.e., cystines are first converted to more stable half-cystines prior to analysis. We present a study which demonstrates that the cystine content can be directly and accurately analyzed at low picomole to femtomole levels. The method involves (a) the employment of a vacuum during sample hydrolysis which permits quantitative recovery of cystine, and (b) the dabsyl chloride precolumn derivatization method which yields stable DABS-cystine that is subsequently analyzed by HPLC. Direct analysis of the cystine residue is important in numerous areas of protein research. It allows, by a single analysis, simultaneous determination of the sulfhydryl (cysteine, as S-carboxymethyl derivative) and disulfide (cystine) contents. The technique can be used to follow the refolding process of fully reduced, cystine-containing proteins. Direct analysis of the cystine content also serves to monitor the extent of inactivation of cystine-containing proteins caused by alkaline pH and heat treatments. In this mode of protein inactivation, cystines are selectively destroyed and converted to lanthionine and lysinoalanine. Both the decrease of cystine and the recoveries of lanthionine and lysinoalanine can be simultaneously evaluated by the proposed method. Examples of these applications are presented here.

A photoaffinity ligand of the acetylcholine-binding site predominantly labels the region 179–207 of the α-subunit on native acetylcholine receptor from Torpedo marmorata

Regions of the Torpedo marmorata acetylcholine receptor (AChR) α‐subunit involved in the binding of acetylcholine were probed with two different covalent ligands. The sulfhydryl‐directed affinity reagent 4‐(N‐maleimido)phenyltrimethylammonium iodide labeled a single α‐subunit cyanogen bromide fragment on the reduced AChR which was identified as α 179–207. The novel photoaffinity ligand p‐(N,N‐dimethylamino)‐benzenediazonium fluoroborate, on the other hand, labeled three distinct α‐chain cyanogen bromide fragments on the unmodified AChR in a carbamylcholine‐protectable manner. The major radiolabeled species was purified and identified by sequence analysis as α 179–207. The acetylcholine‐binding site on the native AChR may thus involve several distinct portions of the α‐chain, with the region α 179–207 making a major contribution to the site.

Sequence determination of eglin c using combined microtechniques of amino acid analysis, peptide isolation, and automatic Edman degradation

The complete amino acid sequence of a proteinase inhibitor, eglin c (Mr 8100), has been determined with less than 150 micrograms of the protein using the following microtechniques: (a) amino acid analysis with a low-nanogram amount of protein hydrolysate using dimethylaminoazobenzene sulfonyl chloride, (b) peptide isolation at the picomole level using the dimethylaminoazobenzene isothiocyanate (DABITC) precolumn derivatization method, and (c) automatic Edman degradation. One amino acid residue has been corrected for the previously reported sequence. The Contribution of each technique to the microsequencing is discussed. In addition, a new high-performance liquid chromatography system that gives a complete baseline separation of all phenylthiohydantoin-amino acids is described.

The structural elements of hirudin which bind to the fibrinogen recognition site of thrombin are exclusively located within its acidic C-terminal tail

Six lysyl residues of human thrombin (LysB21, LysB52, LysB65, LysB106, LysB107 and LysB154) have been previously shown to participate in the binding site of hirudin, a thrombin‐specific inhibitor [(1989) J. Biol. Chem. 264, 7141‐7146]. In this report, we attempted to delineate the region of hirudin which binds to these basic amino acids of thrombin. Using the N‐terminal core domains (r‐Hir1–43 and r‐Hir1–52) derived from recombinant hirudins and synthetic C‐terminal peptides (Hir45–65 and Hir52–65) ‐ all fragments form complexes with thrombin — we are able to demonstrate that the structural elements of hirudin which account for the shielding of these 6 lysyl residues are exclusively located within the acidic C‐terminal region. Since hirudin C‐terminal peptides were shown to bind to a non‐catalytic site of thrombin and inhibit its interaction with fibrinogen [(1987) FEBS Lett. 211, 10‐16], our data consequently imply that these 6 lysyl residues are constituents of the fibrinogen recognition site of thrombin.

The complete amino-acid sequence of anglerfish somatostatin-28 II: A new octacosapeptide containing the (Tyr7, Gly10) derivative of somatostation-14 I*

A new somatostatin‐28 has been isolated from the Teleostean fish (Lophius piscatorius) Brockmann organs. Determination of its aminoacid sequence indicates that it corresponds to an octacosapeptide containing in its C‐terminal end the Tyr‐7 Gly‐10 derivative of somatostatin‐14 I. This structure is in agreement with the one predicted by Hobart et al. (Nature (1980) 288, 137‐141) from a cDNA nucleotide sequence. It demonstrates that, since the corresponding somatostatin‐14 II cannot be detected in this organ, S‐28 II is a terminal product of prosomatostatin II processing in anglerfish pancreatic islets.

Preparation and characterization of proteolyzed forms of human α-thrombin

Abstract The kinetics of the tryptic digestion of human α-thrombin were studied. Based on the results of these studies a procedure for the preparation of highly purified, active human β-thrombin was developed. This β-thrombin contained less than 5% of other thrombin forms, was active towards tripeptidyl paranitroanilide substrates, but had lost more than 99% of its fibrinogen cleaving activity. Protein-chemical characterization of β-thrombin showed that it had been cleaved at a single site (Arg 73 -Asn 74 ) in the B-chain, in contrast to human β-thrombin obtained by autolysis, which is cleaved at both Arg-62 and Arg-73.