scholarly journals Rat α-foetoprotein. Purification, physicochemical characterization, oestrogen-binding properties and chemical modification of the thiol group

1978 ◽  
Vol 175 (1) ◽  
pp. 73-81 ◽  
Author(s):  
Vera Versée ◽  
André O. Barel
Keyword(s):  
Chemical Modification ◽  
Binding Site ◽  
Physicochemical Characterization ◽  
Thiol Group ◽  
Iodoacetic Acid ◽  
Cysteine Residue ◽  
Sulphonic Acid ◽  
Electrophoretic Variants ◽  
Thiol Reagent ◽  
Displacement Experiments

1. Rat α-foetoprotein, an oestrogen-binding foetal globulin, was isolated in large quantities from amniotic fluid and serum by preparative electrophoresis on polyacrylamide slab gels or by chromatography on an immunoadsorbent column. Subsequently the two electrophoretic forms of this protein were separated by electrophoresis on the same medium. 2. Both forms were found to show identical binding with oestradiol. From the extrinsic fluorescence of the bound dye 8-anilinonaphthalene-1-sulphonic acid it was shown that the polarity of the binding site is practically identical for both forms. One residue of tryptophan was determined for both forms. The two electrophoretic variants display the same amount of secondary structure as demonstrated by circular dichroism. 3. The affinity of total α-foetoprotein for oestradiol as a function of pH was studied by using a Sephadex G-25 gel-equilibration method. Maximal binding occurred at pH8.5. Only a fractional number of binding sites per molecule could be measured at pH7.4, whereas at higher pH the number of sites was very close to unity. There was no significant effect of pH on the value of the association constant (Ka=4.3×107±1.2×107m−1). 4. Displacement experiments of bound labelled oestradiol with various steroids have permitted investigation of the specificity of α-foetoprotein. This foetal globulin binds rather strongly compounds that display the rigid structure of the oestratriene skeleton (oestradiol, oestrone). Diminished binding for diethylstilboestrol and a diethylstilboestrol affinity label was observed. No binding was measured with a more flexible structure such as hexoestrol [4,4′-(1,2-diethylethane-1,2-diyl)bisphenol]. 5. Chemical modification of cysteine residues of α-foetoprotein with two alkylating reagents [iodoacetic acid and 8-[N-(iodoacetylaminoethyl)amino]naphthalene-1-sulphonic acid] has very little effect on the oestrogen binding. It is suggested that the oestrogen-binding site does not contain a cysteine residue. From the kinetics of alkylation and from the fluorescence properties of the chemically bound thiol reagent 8-[N-(iodoacetylaminoethyl)amino]naphthalene-1-sulphonic acid], it was demonstrated that the very-slow-reacting thiol group is probably located in a non-polar region of the molecule.

scholarly journals Structural and functional effects of selective chemical modifications of Scapharca inaequivalvis haemoglobins in relation to their unique assembly

1987 ◽  
Vol 241 (2) ◽  
pp. 499-504 ◽  
Author(s):  
A Boffi ◽  
M Gattoni ◽  
R Santucci ◽  
P Vecchini ◽  
F Ascoli ◽  
...  
Keyword(s):  
Functional Properties ◽  
Oxygen Affinity ◽  
Thiol Group ◽  
Cysteine Residue ◽  
Oxygen Binding ◽  
Thiol Groups ◽  
Functional Roles ◽  
Dimeric Unit ◽  
Structural And Functional Properties ◽  
Thiol Reagent

The structural and functional roles of lysyl and thiol groups in the dimeric (HbI) and tetrameric (HbII) haemoglobins from the mollusc Scapharca inaequivalvis have been assessed. In these haemoglobins a unique mode of assembly (the haem-carrying E and F helices form the intersubunit contact of the dimeric unit) is associated with co-operative oxygen binding. Extensive acylation is accompanied by significant haem oxidation. Modification of one or two lysyl residues per chain (corresponding to approximately 20% of the total residues) does not affect the structural and functional properties of both haemoglobins, in line with the proposal that the intersubunit contacts are rich in hydrophobic residues. The modification of the thiol groups does not influence the state of association in both HbI and HbII, despite the location of the cysteine residue common to all polypeptide chains in the vicinity of the major intersubunit contact. The effect on the functional properties depends on the size of the thiol reagent: p-chloromercuribenzoate and phenylmercuric acetate increase the oxygen affinity about 20-fold, but iodoacetamide and mercuric chloride have no effect. Moreover, electrophoresis experiments indicate that p-chloromercuribenzoate is bound in a co-operative fashion, the degree of co-operativity being much higher in the dimeric HbI. Thus, only in HbII are intermediates containing substoichiometric amounts of p-chloromercuribenzoate formed in significant amounts. Their oxygen binding properties show that reaction of only one thiol group/tetramer suffices to alter the oxygen affinity of the molecule.

scholarly journals 13C-n.m.r. of the cyanylated apoflavodoxin and flavodoxin from Clostridium pasteurianum

1993 ◽  
Vol 294 (1) ◽  
pp. 215-218 ◽  
Author(s):  
G M Doherty ◽  
S G Mayhew ◽  
J P G Malthouse
Keyword(s):  
Hydrogen Bonding ◽  
Chemical Shift ◽  
Chemical Modification ◽  
Dissociation Constant ◽  
Chemical Shifts ◽  
Thiol Group ◽  
Cysteine Residue ◽  
Single Step ◽  
Clostridium Pasteurianum ◽  
Megasphaera Elsdenii

The thiol group of the flavodoxin from Clostridium pasteurianum has been cyanylated in a single step using [cyanato-13C]2-nitro-5-thiocyanatobenzoic acid. This chemical modification increases the dissociation constant of the apoflavodoxin-FMN complex 10-fold from 0.33 +/- 0.15 nM to 2.9 +/- 1.3 nM. The thiocyanate carbons of the cyanylated cysteine residue of apoflavodoxin and flavodoxin had chemical shift values of 114.7 and 112.3 p.p.m. respectively. From these chemical shifts we conclude that the binding of FMN by the cyanylated apoflavodoxin causes a decrease in the polarity and/or hydrogen bonding capacity of the environment of the thiocyanate group. We compare these results with those obtained from similar studies on the cyanylated apoflavodoxin and flavodoxin from Megasphaera elsdenii and we discuss how FMN binding and cyanylation perturb the structures of apoflavodoxins from Megasphaera elsdenii and Clostridium pasteurianum.

scholarly journals Chemical modification of serine at the active site of penicillin acylase from Kluyvera citrophila

1991 ◽  
Vol 280 (3) ◽  
pp. 659-662 ◽  
Author(s):  
J Martín ◽  
A Slade ◽  
A Aitken ◽  
R Arche ◽  
R Virden
Keyword(s):  
Active Site ◽  
Tertiary Structure ◽  
Thiol Group ◽  
Iodoacetic Acid ◽  
Penicillin Acylase ◽  
Protein Product ◽  
Side Chain ◽  
Serine Residue ◽  
Conserved Sequence ◽  
Ester Substrate

The site of reaction of penicillin acylase from Kluyvera citrophila with the potent inhibitor phenylmethanesulphonyl fluoride was investigated by incubating the inactivated enzyme with thioacetic acid to convert the side chain of the putative active-site serine residue to that of cysteine. The protein product contained one thiol group, which was reactive towards 2,2′-dipyridyl disulphide and iodoacetic acid. Carboxymethylcysteine was identified as the N-terminal residue of the beta-subunit of the carboxy[3H]methylthiol-protein. No significant changes in tertiary structure were detected in the modified penicillin acylase using near-u.v. c.d. spectroscopy. However, the catalytic activity (kcat) with either an anilide or an ester substrate was decreased in the thiol-protein by a factor of more than 10(4). A comparison of sequences of apparently related acylases shows no other extensive regions of conserved sequence containing an invariant serine residue. The side chain of this residue is proposed as a candidate nucleophile in the formation of an acyl-enzyme during catalysis.

Capturing of the free cysteine residue in the ligand-binding site by affinity labeling of the ORL1 nociceptin receptor

2011 ◽  
Vol 19 (24) ◽  
pp. 7597-7602 ◽  
Author(s):  
Ayami Matsushima ◽  
Hirokazu Nishimura ◽  
Shogo Inamine ◽  
Shiho Uemura ◽  
Yasuyuki Shimohigashi
Keyword(s):  
Ligand Binding ◽  
Binding Site ◽  
Cysteine Residue ◽  
Affinity Labeling ◽  
Ligand Binding Site ◽  
Nociceptin Receptor

scholarly journals Production and comparison of mature single-domain ‘trefoil’ peptides pNR-2/pS2 Cys58 and pNR-2/pS2 Ser58

1995 ◽  
Vol 308 (3) ◽  
pp. 1001-1007 ◽  
Author(s):  
M P Chadwick ◽  
F E B May ◽  
B R Westley
Keyword(s):  
Breast Cancer ◽  
Cancer Cells ◽  
Breast Cancer Cells ◽  
Gel Filtration ◽  
Thiol Group ◽  
Cysteine Residue ◽  
Exchange Chromatography ◽  
Single Domain ◽  
Trefoil Peptides ◽  
Trefoil Peptide

The preparation and purification of recombinant mature pNR-2/pS2, a single-domain member of the ‘trefoil’ family of cysteine-rich secreted proteins, is described. Analysis of recombinant pNR-2/pS2 by ion-exchange chromatography showed that it was heterogeneous. The heterogeneity was reduced by treatment with thiol-group-containing reagents, suggesting that it is caused by the odd number of cysteine residues in mature pNR-2/pS2, and this view was reinforced by mutation of the extra-trefoil domain cysteine residue, Cys58, to a serine residue. Electrophoresis of recombinant pNR-2/pS2 Cys58 and pNR-2/pS2 Ser58 proteins under non-denaturing conditions confirmed that the Ser58 mutant is much more homogeneous, and showed that most of pNR-2/pS2 Ser58 co-migrates as a single band with pNR-2/pS2 secreted from breast-cancer cells in culture. Treatment of recombinant pNR-2/pS2 proteins with various thiol-group-reactive reagents indicated that cysteine is the most effective at producing recombinant pNR-2/pS2 that co-migrates with pNR-2/pS2 secreted by breast-cancer cells. Dithiothreitol appeared to denature the proteins, and GSH was relatively ineffective. pNR-2/pS2 Cys58 treated with cysteine and untreated pNR-2/pS2 Ser58 had the same apparent molecular mass, measured by gel filtration, as pNR-2/pS2 secreted from breast-cancer cells. This is the first report of the production of a recombinant mature single-domain trefoil peptide and should greatly facilitate elucidation of the structure and function of pNR-2/pS2.

scholarly journals The nature of the binding site for aromatic compounds in glycogen phosphorylase b

1975 ◽  
Vol 147 (2) ◽  
pp. 369-371 ◽  
Author(s):  
G Soman ◽  
G Philip
Keyword(s):  
Chemical Modification ◽  
Binding Site ◽  
Aromatic Compounds ◽  
Glycogen Phosphorylase ◽  
Relative Effectiveness ◽  
Rabbit Muscle ◽  
Substituent Constant ◽  
Chemically Modified ◽  
Glycogen Phosphorylase B ◽  
Hydrophobic Nature

The inhibition of rabbit muscle glycogen phosphorylase b (1,4-alpha-D-glucan--orthophosphate alpha-glucosyltransferase, EC 2.4.1.1) by aromatic compounds was examined with 15 compounds. The relative effectiveness of the inhibitors correlated well with increasing substituent constant, pi, indicating the hydrophobic nature of the binding site. The inhibition was not affected by the ionic-strength variation of the assay mixtures. The results predict that the course of chemical modification of this enzyme and the properties of the derivatives depend on the nature of the reagent and on the incorporated groups. Many of the dissimilar and sometimes contradictory results reported for chemical-modification studies and for chemically modified phosphorylase b are explained by the findings presented in the paper.

scholarly journals The pKa of the catalytic histidine residue of chloramphenicol acetyltransferase

1993 ◽  
Vol 290 (1) ◽  
pp. 15-19 ◽  
Author(s):  
A Lewendon ◽  
W V Shaw
Keyword(s):  
Chemical Modification ◽  
Ph Dependence ◽  
Thiol Group ◽  
Chloramphenicol Acetyltransferase ◽  
Histidine Residue ◽  
Pka Values ◽  
Primary Hydroxy Group ◽  
Pka Value ◽  
Catalytic Role ◽  
Kinetics Of

A catalytically essential histidine residue (His-195) of chloramphenicol acetyltransferase (CAT) acts as a general base in catalysis, abstracting a proton from the primary hydroxy group of chloramphenicol. The pKa of His-195 has been determined from the pH-dependence of chemical modification. Both methyl 4-nitrobenzenesulphonate and iodoacetamide inactivate CAT by irreversible modification of His-195. The kinetics of inactivation by methyl 4-nitrobenzenesulphonate are pseudo-first-order, and the pH-dependence of inactivation yields a pKa value of 6.60. Iodoacetamide inactivation proceeds with second-order kinetics and a pKa value of 6.80. An alternative site of modification at the active site of CAT is the thiol group of Cys-31, a residue which has no catalytic role. On replacement of Cys-31 with alanine (Ala-31 CAT), the pH-dependence of iodoacetamide inactivation gives a pKa value of 6.66. The pKa values derived from chemical-modification experiments directed at His-195 are in agreement with the pKa values of 6.62 and 6.61 determined for wild-type and Ala-31 CAT respectively from the pH-dependence of kcat/Km.

scholarly journals The location of the active-site histidine residue in the primary sequence of papain

1968 ◽  
Vol 108 (5) ◽  
pp. 861-866 ◽  
Author(s):  
S. S. Husain ◽  
G. Lowe
Keyword(s):  
Amino Acid ◽  
Sodium Borohydride ◽  
Active Site ◽  
Iodoacetic Acid ◽  
Cysteine Residue ◽  
Histidine Residue ◽  
Primary Sequence ◽  
Active Site Cysteine ◽  
Active Site Histidine ◽  
Active Site Cysteine Residue

Papain that had been irreversibly inhibited with 1,3-dibromo[2−14C]acetone was reduced with sodium borohydride and carboxymethylated with iodoacetic acid. After digestion with trypsin and α-chymotrypsin the radioactive peptides were purified chromatographically. Their amino acid composition indicated that cysteine-25 and histidine-106 were cross-linked. Since cysteine-25 is known to be the active-site cysteine residue, histidine-106 must be the active-site histidine residue.

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