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
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
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
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
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
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
2JOO 2008-03-18 The NMR Solution Structure of Recombinant RGD-hirudin
3VXF 2013-09-04 X/N Joint refinement of Human alpha-thrombin-Bivalirudin complex PD5
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
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
3D49 2009-04-14 1.5 Thrombin Inhibition
3EQ0 2009-08-18 1.53 Thrombin Inhibitor
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
3DUX 2009-06-23 1.6 Understanding Thrombin Inhibition
2ZGB 2008-12-16 1.6 Thrombin Inhibition
1AHT 1996-03-17 1.6 CRYSTAL STRUCTURE OF HUMAN ALPHA-THROMBIN COMPLEXED WITH HIRUGEN AND P-AMIDINOPHENYLPYRUVATE AT 1.6 ANGSTROMS RESOLUTION
2BDY 2006-10-24 1.61 thrombin in complex with inhibitor
4LXB 2014-06-11 1.61 Crystal Structure Analysis of thrombin in complex with compound D58
2ZI2 2009-01-13 1.65 Thrombin Inhibition
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
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
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
1C1U 2000-07-26 1.75 RECRUITING ZINC TO MEDIATE POTENT, SPECIFIC INHIBITION OF SERINE PROTEASES
1O5G 2004-09-21 1.75 Dissecting and Designing Inhibitor Selectivity Determinants at the S1 site Using an Artificial Ala190 Protease (Ala190 uPA)
1GHW 2002-01-22 1.75 A NOVEL SERINE PROTEASE INHIBITION MOTIF INVOLVING A MULTI-CENTERED SHORT HYDROGEN BONDING NETWORK AT THE ACTIVE SITE
1H8I 2001-03-02 1.75 X-ray crystal structure of human alpha-thrombin with a tripeptide phosphonate inhibitor.
1YPK 2006-01-17 1.78 Thrombin Inhibitor Complex
1YPJ 2006-01-17 1.78 Thrombin Inhibitor Complex
2V3O 2008-06-24 1.79 Thrombin with 3-cycle with F
2CF9 2006-06-14 1.79 Complex of recombinant human thrombin with an inhibitor
2V3H 2008-06-24 1.79 Thrombin with 3-cycle no F
1YPG 2006-01-17 1.8 Thrombin Inhibitor Complex
1BA8 1999-04-27 1.8 THROMBIN INHIBITOR WITH A RIGID TRIPEPTIDYL ALDEHYDES
1AY6 1998-03-18 1.8 THROMBIN INHIBITOR FROM THEONALLA, CYCLOTHEANAMIDE-BASED MACROCYCLIC TRIPEPTIDE MOTIF
2ZNK 2009-04-14 1.8 Thrombin Inhibition
1Z71 2005-05-17 1.8 thrombin and P2 pyridine N-oxide inhibitor complex structure
1A3B 1998-06-03 1.8 COMPLEX OF HUMAN ALPHA-THROMBIN WITH THE BIFUNCTIONAL BORONATE INHIBITOR BOROLOG1
2ZGX 2008-12-16 1.8 Thrombin Inhibition
1NM6 2003-09-02 1.8 thrombin in complex with selective macrocyclic inhibitor at 1.8A
1QBV 1999-10-27 1.8 CRYSTAL STRUCTURE OF THROMBIN COMPLEXED WITH AN GUANIDINE-MIMETIC INHIBITOR
1SL3 2004-08-03 1.81 crystal structue of Thrombin in complex with a potent P1 heterocycle-Aryl based 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
3C1K 2008-03-18 1.84 Crystal structure of thrombin in complex with inhibitor 15
2PW8 2007-08-21 1.84 Crystal structure of sulfo-hirudin complexed to thrombin
1A3E 1998-06-03 1.85 COMPLEX OF HUMAN ALPHA-THROMBIN WITH THE BIFUNCTIONAL BORONATE INHIBITOR BOROLOG2
1YPM 2006-01-17 1.85 X-ray crystal structure of thrombin inhibited by synthetic cyanopeptide analogue RA-1014
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
1YPL 2006-01-17 1.85 X-ray crystal structure of thrombin inhibited by synthetic cyanopeptide analogue RA-1008
1GHV 2002-01-22 1.85 A NOVEL SERINE PROTEASE INHIBITION MOTIF INVOLVING A MULTI-CENTERED SHORT HYDROGEN BONDING NETWORK AT THE ACTIVE SITE
1GHY 2002-01-22 1.85 A NOVEL SERINE PROTEASE INHIBITION MOTIF INVOLVING A MULTI-CENTERED SHORT HYDROGEN BONDING NETWORK AT THE ACTIVE SITE
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
1VR1 1999-12-29 1.9 Specifity for Plasminogen Activator Inhibitor-1
4AX9 2012-06-20 1.9 Human thrombin complexed with Napsagatran, RO0466240
1TA6 2004-06-08 1.9 Crystal structure of thrombin in complex with compound 14b
1G32 2001-10-23 1.9 THROMBIN INHIBITOR COMPLEX
1NO9 2003-08-26 1.9 Design of weakly basic thrombin inhibitors incorporating novel P1 binding functions: molecular and X-ray crystallographic studies.
1ZRB 2005-06-07 1.9 Thrombin in complex with an azafluorenyl inhibitor 23b
1C5O 2000-12-22 1.9 STRUCTURAL BASIS FOR SELECTIVITY OF A SMALL MOLECULE, S1-BINDING, SUB-MICROMOLAR INHIBITOR OF UROKINASE TYPE PLASMINOGEN ACTIVATOR
1C1W 2000-07-26 1.9 RECRUITING ZINC TO MEDIATE POTENT, SPECIFIC INHIBITION OF SERINE PROTEASES
1SB1 2004-06-08 1.9 Novel Non-Covalent Thrombin Inhibitors Incorporating P1 4,5,6,7-Tetrahydrobenzothiazole Arginine Side Chain Mimetics
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
1MU8 2004-04-06 2.0 thrombin-hirugen_l-378,650
1AE8 1997-12-03 2.0 HUMAN ALPHA-THROMBIN INHIBITION BY EOC-D-PHE-PRO-AZALYS-ONP
1G30 2001-10-23 2.0 THROMBIN INHIBITOR COMPLEX
1AFE 1997-12-03 2.0 HUMAN ALPHA-THROMBIN INHIBITION BY CBZ-PRO-AZALYS-ONP
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
1QJ1 2000-06-22 2.0 Novel Covalent Active Site Thrombin Inhibitors
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
1UMA 1996-11-08 2.0 ALPHA-THROMBIN (HIRUGEN) COMPLEXED WITH NA-(N,N-DIMETHYLCARBAMOYL)-ALPHA-AZALYSINE
1AD8 1997-11-12 2.0 COMPLEX OF THROMBIN WITH AND INHIBITOR CONTAINING A NOVEL P1 MOIETY
1NT1 2003-09-02 2.0 thrombin in complex with selective macrocyclic inhibitor
2GDE 2007-03-20 2.0 Thrombin in complex with inhibitor
1BCU 1998-10-14 2.0 ALPHA-THROMBIN COMPLEXED WITH HIRUGEN AND PROFLAVIN
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
1C4U 2000-09-27 2.1 SELECTIVE NON ELECTROPHILIC THROMBIN INHIBITORS WITH CYCLOHEXYL MOIETIES.
1CA8 1999-04-27 2.1 Thrombin inhibitors with rigid tripeptidyl aldehydes
1AIX 1997-10-15 2.1 HUMAN ALPHA-THROMBIN TERNARY COMPLEX WITH EXOSITE INHIBITOR HIRUGEN AND ACTIVE SITE INHIBITOR PHCH2OCO-D-DPA-PRO-BOROVAL
1D3P 2000-10-04 2.1 CRYSTAL STRUCTURE OF HUMAN APLHA-THROMBIN IN COMPLEX WITH BENZO[B]THIOPHENE INHIBITOR 3
1HXF 1997-01-27 2.1 HUMAN THROMBIN COMPLEX WITH HIRUDIN VARIANT
1BB0 1999-04-27 2.1 THROMBIN INHIBITORS WITH RIGID TRIPEPTIDYL ALDEHYDES
1HXE 1996-11-08 2.1 SERINE PROTEASE
1KTT 2002-02-06 2.1 Thrombin inhibitor complex
2ZF0 2008-11-04 2.2 Exploring Thrombin S1 Pocket
1QJ7 2000-06-22 2.2 Novel Covalent Active Site 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
1QJ6 2000-06-22 2.2 Novel Covalent Active Site Thrombin Inhibitors
1ZGI 2005-09-27 2.2 thrombin in complex with an oxazolopyridine inhibitor 21
1ZGV 2005-09-27 2.2 Thrombin in complex with an oxazolopyridine inhibitor 2
4MLF 2013-09-25 2.2 Crystal structure for the complex of thrombin mutant D102N and hirudin
1QHR 2000-05-31 2.2 NOVEL COVALENT ACTIVE SITE THROMBIN INHIBITORS
3EGK 2008-09-30 2.2 KNOBLE Inhibitor
2HGT 1994-01-31 2.2 STRUCTURE OF THE HIRUGEN AND HIRULOG 1 COMPLEXES OF ALPHA-THROMBIN
1AWF 1998-10-28 2.2 NOVEL COVALENT THROMBIN INHIBITOR FROM PLANT EXTRACT
2ZFP 2008-12-16 2.25 Thrombin Inibition
3LDX 2011-08-10 2.25 Discovery and Clinical Evaluation of RWJ-671818, a Thrombin Inhibitor with an Oxyguanidine P1 Motif
1ABI 1994-01-31 2.3 STRUCTURE OF THE HIRULOG 3-THROMBIN COMPLEX AND NATURE OF THE S' SUBSITES OF SUBSTRATES AND INHIBITORS
1D9I 2000-10-30 2.3 STRUCTURE OF THROMBIN COMPLEXED WITH SELECTIVE NON-ELECTOPHILIC INHIBITORS HAVING CYCLOHEXYL MOIETIES AT P1
1FPC 1995-02-27 2.3 ACTIVE SITE MIMETIC INHIBITION OF THROMBIN
1TA2 2004-06-08 2.3 Crystal structure of thrombin in complex with compound 1
1XM1 2005-05-10 2.3 Nonbasic Thrombin Inhibitor Complex
1TWX 2005-04-19 2.4 Crystal structure of the thrombin mutant D221A/D222K
1KTS 2002-02-06 2.4 Thrombin Inhibitor Complex
3DT0 2009-06-23 2.4 Understanding Thrombin Inhibition
1FPH 1994-01-31 2.5 THE INTERACTION OF THROMBIN WITH FIBRINOGEN: A STRUCTURAL BASIS FOR ITS SPECIFICITY
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
4AZ2 2012-08-15 2.6 Human thrombin - inhibitor complex
4AYV 2012-08-15 2.8 Human thrombin - inhibitor complex
1HRT 1994-01-31 2.8 THE STRUCTURE OF A COMPLEX OF BOVINE ALPHA-THROMBIN AND RECOMBINANT HIRUDIN AT 2.8 ANGSTROMS RESOLUTION
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
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
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
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
98.0 Hirudin variant-1 P84590 HIRUD_POEVI
90.8 Hirudin variant-1 P28504 HIR2_HIRME
92.3 Hirudin variant-1 P28503 HIR2A_HIRME
92.3 Hirudin variant-1 P28506 HIR2B_HIRME
95.4 Hirudin variant-1 P28510 HIR3B_HIRME
93.8 Hirudin variant-1 P28508 HIR3P_HIRME
96.9 Hirudin variant-1 P28509 HIR3_HIRME
96.9 Hirudin variant-1 P28511 HIR3C_HIRME
100.0 Hirudin variant-1 P01050 HIRV1_HIRME