Characterization, stability and refolding of recombinant hirudin.


Abstract

A recombinant variant of hirudin, the blood-clotting inhibitor of the leech Hirudo medicinalis, has been characterized employing spectroscopic and hydrodynamic techniques. Conditions have been defined for efficient reconstitution of the native, disulfide-bonded inhibitor from completely unfolded, reduced polypeptide chains. The spectral properties of the native inhibitor are consistent with previous results on the solution structure of hirudin. Extremely low circular dichroism in the far ultraviolet ([theta]Mr,220 nm = -8 +/- 1 x 10(2) deg.cm2.dmol-1) indicates a very low content of regular secondary structure. Although both tyrosine residues of the recombinant inhibitor titrate around pH 10.6, typical for solvent-exposed tyrosines, fluorescence emission and near-ultraviolet circular dichroism suggest that at least one of the tyrosines is partially shielded from solvent quench, and immobilized in an asymmetric environment. Reversible thermal unfolding of hirudin around 65 degrees C is indicated by the disappearance of its dichroic absorption in the near ultraviolet and by a fourfold increase in ellipticity at 225 nm. The transition can be approximated by a two-state model with a transition enthalpy of delta Hvan't Hoff = 159 kJ/mol and a transition entropy of 464 J.mol-1.K-1. Reduced hirudin at room temperature is largely unfolded and inactive as an inhibitor of thrombin assayed with a low-molecular-mass substrate. Refolding and reoxidation are observed at alkaline pH in the presence of a mixture of glutathione and glutathione disulfide. Spectroscopy, thrombin inhibition, and reversed-phase HPLC indicate reconstitution yields close to 100% and that the reconstituted inhibitor is identical to the native starting material. Study holds ProTherm entries: 7171, 7172 Extra Details: disulfide-bonded inhibitor; secondary structure;,solvent-exposed tyrosine; reversed-phase HPLC

Submission Details

ID: ZZrPCDAF4

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:33 p.m.

Version: 1

Publication Details
Otto A;Seckler R,Eur. J. Biochem. (1991) Characterization, stability and refolding of recombinant hirudin. PMID:1935981
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
4HIR 1990-01-15 SOLUTION STRUCTURE OF RECOMBINANT HIRUDIN AND THE LYS-47 (RIGHT ARROW) GLU MUTANT. A NUCLEAR MAGNETIC RESONANCE AND HYBRID DISTANCE GEOMETRY-DYNAMICAL SIMULATED ANNEALING STUDY
3VXF 2013-09-04 X/N Joint refinement of Human alpha-thrombin-Bivalirudin complex PD5
1HIC 1994-01-31 THE NMR SOLUTION STRUCTURE OF HIRUDIN(1-51) AND COMPARISON WITH CORRESPONDING THREE-DIMENSIONAL STRUCTURES DETERMINED USING THE COMPLETE 65-RESIDUE HIRUDIN POLYPEPTIDE CHAIN
6HIR 1990-01-15 SOLUTION STRUCTURE OF RECOMBINANT HIRUDIN AND THE LYS-47 (RIGHT ARROW) GLU MUTANT. A NUCLEAR MAGNETIC RESONANCE AND HYBRID DISTANCE GEOMETRY-DYNAMICAL SIMULATED ANNEALING STUDY
2JOO 2008-03-18 The NMR Solution Structure of Recombinant RGD-hirudin
2HIR 1990-01-15 SOLUTION STRUCTURE OF RECOMBINANT HIRUDIN AND THE LYS-47 (RIGHT ARROW) GLU MUTANT. A NUCLEAR MAGNETIC RESONANCE AND HYBRID DISTANCE GEOMETRY-DYNAMICAL SIMULATED ANNEALING STUDY
5HIR 1990-01-15 SOLUTION STRUCTURE OF RECOMBINANT HIRUDIN AND THE LYS-47 (RIGHT ARROW) GLU MUTANT. A NUCLEAR MAGNETIC RESONANCE AND HYBRID DISTANCE GEOMETRY-DYNAMICAL SIMULATED ANNEALING STUDY
3VXE 2013-09-04 1.25 Human alpha-thrombin-Bivalirudin complex at PD5.0
2UUF 2007-09-04 1.26 Thrombin-hirugen binary complex at 1.26A resolution
2CF8 2006-06-14 1.3 Complex of recombinant human thrombin with an inhibitor
2UUJ 2007-09-04 1.32 Thrombin-hirugen-gw473178 ternary complex at 1.32A resolution
2UUK 2007-09-04 1.39 Thrombin-hirugen-gw420128 ternary complex at 1.39A resolution
1H8D 2001-02-26 1.4 X-ray structure of the human alpha-thrombin complex with a tripeptide phosphonate inhibitor.
1C5L 2000-12-22 1.47 STRUCTURAL BASIS FOR SELECTIVITY OF A SMALL MOLECULE, S1-BINDING, SUB-MICROMOLAR INHIBITOR OF UROKINASE TYPE PLASMINOGEN ACTIVATOR
2ZFF 2008-12-16 1.47 Exploring Thrombin S1-pocket
3D49 2009-04-14 1.5 Thrombin Inhibition
4YES 2015-06-17 1.5 Thrombin in complex with (S)-(4-chloro-2-((1-(5-methyl-1H-pyrrole-2-carbonyl)pyrrolidine-2-carboxamido)methyl)phenyl)methanaminium
1C5N 2000-12-22 1.5 STRUCTURAL BASIS FOR SELECTIVITY OF A SMALL MOLECULE, S1-BINDING, SUB-MICROMOLAR INHIBITOR OF UROKINASE TYPE PLASMINOGEN ACTIVATOR
3EQ0 2009-08-18 1.53 Thrombin Inhibitor
1VZQ 2005-06-20 1.54 Complex of thrombin with designed inhibitor 7165
3UTU 2012-08-29 1.55 High affinity inhibitor of human thrombin
2ZC9 2008-10-28 1.58 Thrombin in complex with Inhibitor
1O2G 2003-05-13 1.58 Elaborate Manifold of Short Hydrogen Bond Arrays Mediating Binding of Active Site-Directed Serine Protease Inhibitors
1AHT 1996-03-17 1.6 CRYSTAL STRUCTURE OF HUMAN ALPHA-THROMBIN COMPLEXED WITH HIRUGEN AND P-AMIDINOPHENYLPYRUVATE AT 1.6 ANGSTROMS RESOLUTION
3DUX 2009-06-23 1.6 Understanding Thrombin Inhibition
2ZGB 2008-12-16 1.6 Thrombin Inhibition
4LXB 2014-06-11 1.61 Crystal Structure Analysis of thrombin in complex with compound D58
2BDY 2006-10-24 1.61 thrombin in complex with inhibitor
2ZIQ 2009-01-13 1.65 Thrombin Inhibition
1GHX 2002-01-22 1.65 A NOVEL SERINE PROTEASE INHIBITION MOTIF INVOLVING A MULTI-CENTERED SHORT HYDROGEN BONDING NETWORK AT THE ACTIVE SITE
2ZI2 2009-01-13 1.65 Thrombin Inhibition
1OYT 2003-06-24 1.67 COMPLEX OF RECOMBINANT HUMAN THROMBIN WITH A DESIGNED FLUORINATED INHIBITOR
2ZO3 2009-04-14 1.7 Bisphenylic Thrombin Inhibitors
1DOJ 2000-11-03 1.7 Crystal structure of human alpha-thrombin*RWJ-51438 complex at 1.7 A
2ZDV 2008-10-28 1.72 Exploring Thrombin S1 pocket
2ZDA 2008-10-28 1.73 Exploring Thrombin S1 pocket
1GJ5 2002-04-27 1.73 SELECTIVITY AT S1, H2O DISPLACEMENT, UPA, TPA, SER190/ALA190 PROTEASE, STRUCTURE-BASED DRUG DESIGN
3DHK 2009-05-19 1.73 Bisphenylic Thrombin Inhibitors
1GHW 2002-01-22 1.75 A NOVEL SERINE PROTEASE INHIBITION MOTIF INVOLVING A MULTI-CENTERED SHORT HYDROGEN BONDING NETWORK AT THE ACTIVE SITE
1O5G 2004-09-21 1.75 Dissecting and Designing Inhibitor Selectivity Determinants at the S1 site Using an Artificial Ala190 Protease (Ala190 uPA)
1H8I 2001-03-02 1.75 X-ray crystal structure of human alpha-thrombin with a tripeptide phosphonate inhibitor.
1C1U 2000-07-26 1.75 RECRUITING ZINC TO MEDIATE POTENT, SPECIFIC INHIBITION OF SERINE PROTEASES
1YPK 2006-01-17 1.78 Thrombin Inhibitor Complex
1YPJ 2006-01-17 1.78 Thrombin Inhibitor Complex
2CF9 2006-06-14 1.79 Complex of recombinant human thrombin with an inhibitor
2V3O 2008-06-24 1.79 Thrombin with 3-cycle with F
2V3H 2008-06-24 1.79 Thrombin with 3-cycle no F
1AY6 1998-03-18 1.8 THROMBIN INHIBITOR FROM THEONALLA, CYCLOTHEANAMIDE-BASED MACROCYCLIC TRIPEPTIDE MOTIF
1YPG 2006-01-17 1.8 Thrombin Inhibitor Complex
2ZGX 2008-12-16 1.8 Thrombin Inhibition
2ZNK 2009-04-14 1.8 Thrombin Inhibition
1NM6 2003-09-02 1.8 thrombin in complex with selective macrocyclic inhibitor at 1.8A
1A3B 1998-06-03 1.8 COMPLEX OF HUMAN ALPHA-THROMBIN WITH THE BIFUNCTIONAL BORONATE INHIBITOR BOROLOG1
1Z71 2005-05-17 1.8 thrombin and P2 pyridine N-oxide inhibitor complex structure
1BA8 1999-04-27 1.8 THROMBIN INHIBITOR WITH A RIGID TRIPEPTIDYL ALDEHYDES
1QBV 1999-10-27 1.8 CRYSTAL STRUCTURE OF THROMBIN COMPLEXED WITH AN GUANIDINE-MIMETIC INHIBITOR
1GJ4 2002-04-27 1.81 SELECTIVITY AT S1, H2O DISPLACEMENT, UPA, TPA, SER190/ALA190 PROTEASE, STRUCTURE-BASED DRUG DESIGN
1YPE 2006-01-17 1.81 Thrombin Inhibitor Complex
1SL3 2004-08-03 1.81 crystal structue of Thrombin in complex with a potent P1 heterocycle-Aryl based inhibitor
2PW8 2007-08-21 1.84 Crystal structure of sulfo-hirudin complexed to thrombin
3C1K 2008-03-18 1.84 Crystal structure of thrombin in complex with inhibitor 15
1GHY 2002-01-22 1.85 A NOVEL SERINE PROTEASE INHIBITION MOTIF INVOLVING A MULTI-CENTERED SHORT HYDROGEN BONDING NETWORK AT THE ACTIVE SITE
1AI8 1997-10-15 1.85 HUMAN ALPHA-THROMBIN TERNARY COMPLEX WITH THE EXOSITE INHIBITOR HIRUGEN AND ACTIVE SITE INHIBITOR PHCH2OCO-D-DPA-PRO-BOROMPG
1YPM 2006-01-17 1.85 X-ray crystal structure of thrombin inhibited by synthetic cyanopeptide analogue RA-1014
1A3E 1998-06-03 1.85 COMPLEX OF HUMAN ALPHA-THROMBIN WITH THE BIFUNCTIONAL BORONATE INHIBITOR BOROLOG2
1GHV 2002-01-22 1.85 A NOVEL SERINE PROTEASE INHIBITION MOTIF INVOLVING A MULTI-CENTERED SHORT HYDROGEN BONDING NETWORK AT THE ACTIVE SITE
1YPL 2006-01-17 1.85 X-ray crystal structure of thrombin inhibited by synthetic cyanopeptide analogue RA-1008
4BAN 2013-01-16 1.87 Thrombin in complex with inhibitor
4BAM 2013-01-16 1.88 Thrombin in complex with inhibitor
4BAQ 2013-01-16 1.89 Thrombin in complex with inhibitor
1C5O 2000-12-22 1.9 STRUCTURAL BASIS FOR SELECTIVITY OF A SMALL MOLECULE, S1-BINDING, SUB-MICROMOLAR INHIBITOR OF UROKINASE TYPE PLASMINOGEN ACTIVATOR
1TA6 2004-06-08 1.9 Crystal structure of thrombin in complex with compound 14b
1SB1 2004-06-08 1.9 Novel Non-Covalent Thrombin Inhibitors Incorporating P1 4,5,6,7-Tetrahydrobenzothiazole Arginine Side Chain Mimetics
1ZRB 2005-06-07 1.9 Thrombin in complex with an azafluorenyl inhibitor 23b
4AX9 2012-06-20 1.9 Human thrombin complexed with Napsagatran, RO0466240
1NO9 2003-08-26 1.9 Design of weakly basic thrombin inhibitors incorporating novel P1 binding functions: molecular and X-ray crystallographic studies.
1G32 2001-10-23 1.9 THROMBIN INHIBITOR COMPLEX
1VR1 1999-12-29 1.9 Specifity for Plasminogen Activator Inhibitor-1
1C1W 2000-07-26 1.9 RECRUITING ZINC TO MEDIATE POTENT, SPECIFIC INHIBITION OF SERINE PROTEASES
4BAH 2013-01-16 1.94 Thrombin in complex with inhibitor
4BAK 2013-01-16 1.94 Thrombin in complex with inhibitor
2ZHQ 2009-01-13 1.96 Thrombin Inhibition
1C1V 2000-07-26 1.98 RECRUITING ZINC TO MEDIATE POTENT, SPECIFIC INHIBITION OF SERINE PROTEASES
1MU6 2004-04-06 1.99 Crystal Structure of Thrombin in Complex with L-378,622
1T4U 2005-03-22 2.0 Crystal Structure Analysis of a novel Oxyguanidine bound to Thrombin
1AD8 1997-11-12 2.0 COMPLEX OF THROMBIN WITH AND INHIBITOR CONTAINING A NOVEL P1 MOIETY
1AE8 1997-12-03 2.0 HUMAN ALPHA-THROMBIN INHIBITION BY EOC-D-PHE-PRO-AZALYS-ONP
1UMA 1996-11-08 2.0 ALPHA-THROMBIN (HIRUGEN) COMPLEXED WITH NA-(N,N-DIMETHYLCARBAMOYL)-ALPHA-AZALYSINE
2GDE 2007-03-20 2.0 Thrombin in complex with inhibitor
1QJ1 2000-06-22 2.0 Novel Covalent Active Site Thrombin Inhibitors
1T4V 2005-03-22 2.0 Crystal Structure Analysis of a novel Oxyguanidine bound to Thrombin
1IHS 1994-01-31 2.0 CRYSTAL STRUCTURE OF THE COMPLEX OF HUMAN ALPHA-THROMBIN AND NON-HYDROLYZABLE BIFUNCTIONAL INHIBITORS, HIRUTONIN-2 AND HIRUTONIN-6
1G30 2001-10-23 2.0 THROMBIN INHIBITOR COMPLEX
1D6W 2000-10-25 2.0 STRUCTURE OF THROMBIN COMPLEXED WITH SELECTIVE NON-ELECTROPHILIC INHIBITORS HAVING CYCLOHEXYL MOIETIES AT P1
1MUE 2004-04-06 2.0 Thrombin-Hirugen-L405,426
1BCU 1998-10-14 2.0 ALPHA-THROMBIN COMPLEXED WITH HIRUGEN AND PROFLAVIN
1NT1 2003-09-02 2.0 thrombin in complex with selective macrocyclic inhibitor
1MU8 2004-04-06 2.0 thrombin-hirugen_l-378,650
1AFE 1997-12-03 2.0 HUMAN ALPHA-THROMBIN INHIBITION BY CBZ-PRO-AZALYS-ONP
1WAY 2005-01-27 2.02 Active site thrombin inhibitors
1RIW 2004-11-09 2.04 Thrombin in complex with natural product inhibitor Oscillarin
1D3D 2000-10-04 2.04 CRYSTAL STRUCTURE OF HUMAN ALPHA THROMBIN IN COMPLEX WITH BENZOTHIOPHENE INHIBITOR 4
1D4P 1999-10-20 2.07 CRYSTAL STRUCTURE OF HUMAN ALPHA THROMBIN IN COMPLEX WITH 5-AMIDINOINDOLE-4-BENZYLPIPERIDINE INHIBITOR
1KTT 2002-02-06 2.1 Thrombin inhibitor complex
1C4U 2000-09-27 2.1 SELECTIVE NON ELECTROPHILIC THROMBIN INHIBITORS WITH CYCLOHEXYL MOIETIES.
1BB0 1999-04-27 2.1 THROMBIN INHIBITORS WITH RIGID TRIPEPTIDYL ALDEHYDES
1CA8 1999-04-27 2.1 Thrombin inhibitors with rigid tripeptidyl aldehydes
1HXE 1996-11-08 2.1 SERINE PROTEASE
1HXF 1997-01-27 2.1 HUMAN THROMBIN COMPLEX WITH HIRUDIN VARIANT
1D3P 2000-10-04 2.1 CRYSTAL STRUCTURE OF HUMAN APLHA-THROMBIN IN COMPLEX WITH BENZO[B]THIOPHENE INHIBITOR 3
3C27 2009-02-17 2.18 Cyanofluorophenylacetamides as Orally Efficacious Thrombin Inhibitors
1TMT 1994-09-30 2.2 CHANGES IN INTERACTIONS IN COMPLEXES OF HIRUDIN DERIVATIVES AND HUMAN ALPHA-THROMBIN DUE TO DIFFERENT CRYSTAL FORMS
1QJ7 2000-06-22 2.2 Novel Covalent Active Site Thrombin Inhibitors
1QHR 2000-05-31 2.2 NOVEL COVALENT ACTIVE SITE THROMBIN INHIBITORS
3EGK 2008-09-30 2.2 KNOBLE Inhibitor
1AWF 1998-10-28 2.2 NOVEL COVALENT THROMBIN INHIBITOR FROM PLANT EXTRACT
4MLF 2013-09-25 2.2 Crystal structure for the complex of thrombin mutant D102N and hirudin
1ZGV 2005-09-27 2.2 Thrombin in complex with an oxazolopyridine inhibitor 2
2ZF0 2008-11-04 2.2 Exploring Thrombin S1 Pocket
1QJ6 2000-06-22 2.2 Novel Covalent Active Site Thrombin Inhibitors
2HGT 1994-01-31 2.2 STRUCTURE OF THE HIRUGEN AND HIRULOG 1 COMPLEXES OF ALPHA-THROMBIN
1ZGI 2005-09-27 2.2 thrombin in complex with an oxazolopyridine inhibitor 21
3LDX 2011-08-10 2.25 Discovery and Clinical Evaluation of RWJ-671818, a Thrombin Inhibitor with an Oxyguanidine P1 Motif
2ZFP 2008-12-16 2.25 Thrombin Inibition
1FPC 1995-02-27 2.3 ACTIVE SITE MIMETIC INHIBITION OF THROMBIN
1D9I 2000-10-30 2.3 STRUCTURE OF THROMBIN COMPLEXED WITH SELECTIVE NON-ELECTOPHILIC INHIBITORS HAVING CYCLOHEXYL MOIETIES AT P1
1XM1 2005-05-10 2.3 Nonbasic Thrombin Inhibitor Complex
1ABI 1994-01-31 2.3 STRUCTURE OF THE HIRULOG 3-THROMBIN COMPLEX AND NATURE OF THE S' SUBSITES OF SUBSTRATES AND INHIBITORS
1TA2 2004-06-08 2.3 Crystal structure of thrombin in complex with compound 1
1TWX 2005-04-19 2.4 Crystal structure of the thrombin mutant D221A/D222K
3DT0 2009-06-23 2.4 Understanding Thrombin Inhibition
1KTS 2002-02-06 2.4 Thrombin Inhibitor Complex
3TU7 2011-10-12 2.49 Human alpha-thrombin complexed with N-(methylsulfonyl)-D-phenylalanyl-N-((1-carbamimidoyl-4-piperidinyl)methyl)-l-prolinamide (BMS-189664)
1FPH 1994-01-31 2.5 THE INTERACTION OF THROMBIN WITH FIBRINOGEN: A STRUCTURAL BASIS FOR ITS SPECIFICITY
4AZ2 2012-08-15 2.6 Human thrombin - inhibitor complex
1HDT 1995-10-15 2.6 STRUCTURE OF A RETRO-BINDING PEPTIDE INHIBITOR COMPLEXED WITH HUMAN ALPHA-THROMBIN
4AYY 2012-08-15 2.6 Human thrombin - inhibitor complex
1BMM 1996-12-07 2.6 HUMAN ALPHA-THROMBIN COMPLEXED WITH [S-(R*,R*)]-4-[(AMINOIMINOMETHYL)AMINO]-N-[[1-[3-HYDROXY-2-[(2-NAPHTHALENYLSULFONYL)AMINO]-1-OXOPROPYL]-2-PYRROLIDINYL] METHYL]BUTANAMIDE (BMS-186282)
1HRT 1994-01-31 2.8 THE STRUCTURE OF A COMPLEX OF BOVINE ALPHA-THROMBIN AND RECOMBINANT HIRUDIN AT 2.8 ANGSTROMS RESOLUTION
4AYV 2012-08-15 2.8 Human thrombin - inhibitor complex
1BMN 1996-12-07 2.8 HUMAN ALPHA-THROMBIN COMPLEXED WITH [S-(R*,R*)]-1-(AMINOIMINOMETHYL)-N-[[1-[N-[(2-NAPHTHALENYLSULFONYL)-L-SERYL]-PYRROLIDINYL]METHYL]-3-PIPERIDENECARBOXAMIDE (BMS-189090)
1D3Q 2000-10-04 2.9 CRYSTAL STRUCTURE OF HUMAN ALPHA THROMBIN IN COMPLEX WITH BENZO[B]THIOPHENE INHIBITOR 2
1DWD 1994-01-31 3.0 CRYSTALLOGRAPHIC ANALYSIS AT 3.0-ANGSTROMS RESOLUTION OF THE BINDING TO HUMAN THROMBIN OF FOUR ACTIVE SITE-DIRECTED INHIBITORS
1DWC 1994-01-31 3.0 CRYSTALLOGRAPHIC ANALYSIS AT 3.0-ANGSTROMS RESOLUTION OF THE BINDING TO HUMAN THROMBIN OF FOUR ACTIVE SITE-DIRECTED INHIBITORS
1DWE 1994-01-31 3.0 Crystallographic analysis at 3.0-Angstroms resolution of the binding to human thrombin of four active site-directed inhibitors
1D3T 2000-10-04 3.0 CRYSTAL STRUCTURE OF HUMAN ALPHA THROMBIN IN COMPLEX WITH BENZO[B]THIOPHENE INHIBITOR 1
1DWB 1994-01-31 3.16 CRYSTALLOGRAPHIC ANALYSIS AT 3.0-ANGSTROMS RESOLUTION OF THE BINDING TO HUMAN THROMBIN OF FOUR ACTIVE SITE-DIRECTED INHIBITORS

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Hirudin variant-1 P01050 HIRV1_HIRME
96.9 Hirudin variant-1 P28511 HIR3C_HIRME
96.9 Hirudin variant-1 P28509 HIR3_HIRME
93.8 Hirudin variant-1 P28508 HIR3P_HIRME
95.4 Hirudin variant-1 P28510 HIR3B_HIRME
92.3 Hirudin variant-1 P28506 HIR2B_HIRME
92.3 Hirudin variant-1 P28503 HIR2A_HIRME
90.8 Hirudin variant-1 P28504 HIR2_HIRME
98.0 Hirudin variant-1 P84590 HIRUD_POEVI