Terminal NH2 Groups Research Articles

Synthesis and Structure Determinations of Complexes Containing a Five-Membered Lactam Structure Based on Organohydrazido(2−) Ligands1

Acid-catalyzed reactions of the hydrazido(2−) complexes cis,mer-[WX2(NNH2)(PMe2Ph)3] (X = Cl, Br) with phthalaldehyde gave the (phthalimidin-2-yl)imido complexes cis,mer-[WX2(NNCH2C6H4CO)(PMe2Ph)3], via the condensation of the terminal NH2 group with one of the formyl groups and the following cyclization to form a phthalimidine ring. Crystal structure of the chloro complex 3a was unambiguously determined by X-ray analysis. Reaction of 3a with HBr liberated the (phthalimidin-2-yl)imido ligand as 2-aminophthalimidine in moderate yield, while treatment of 3a with KOH in THF selectively cleaved the N−N bond to give phthalimidine. A similar condensation of the hydrazido(2−) complex trans-[WF(NNH2)(dppe)2]+ (8a+; dppe = Ph2PCH2CH2PPh2) with phthalaldehyde resulted in the formation of the diazoalkane complex trans-[WF(NNCHC6H4CHO)(dppe)2]+. However, further treatment of the latter complex with AlCl3 afforded the corresponding (phthalimidin-2-yl)imido complex trans-[WF(NNCH2C6H4CO)(dppe)2]+. When 8a+ and its molybdenum analogue were reacted with 2,5-dimethoxy-2,5-dihydrofuran in the presence of a catalytic amount of acid, trans-[MF(NNCHCHCH2CO)(dppe)2]+ (11+; M = Mo, W) was formed as the kinetic product, which gradually isomerized to the thermodynamically more stable compound trans-[MF(NNCH2CHCHCO)(dppe)2]+ (12+). Both 11+ and 12+ (M = W) were crystallographically characterized, and the mechanism for the isomerization of 11+ to 12+ was proposed based on the results of the 1H NMR measurements.

The Seven-transmembrane-spanning Receptors for Endothelin and Thrombin Cause Proliferation of Airway Smooth Muscle Cells and Activation of the Extracellular Regulated Kinase and c-Jun NH2-terminal Kinase Groups of Mitogen-activated Protein Kinases

In airway smooth muscle cells ligand binding to the seven-transmembrane endothelin and thrombin receptors stimulates cell growth. Rapid activation of the extracellular regulated kinase 2 and c-Jun NH2-terminal kinase groups of mitogen-activated protein kinases was also observed. The results demonstrate a novel mechanism of seven-transmembrane receptor signaling involving activation of the Jun kinase pathway. Receptor coupling to Jun kinase activation may involve heterotrimeric G proteins since the kinase was enzymatically activated in cells treated with aluminum fluoride. The activity of Raf-1, measured by immune complex kinase assay, revealed that platelet-derived growth factor and phorbol 12-myristate 13-acetate both stimulated Raf-1 activity, while thrombin and endothelin did not appreciably stimulate Raf-1. The data suggest that endothelin and thrombin stimulate Raf-1-independent mechanisms of mitogen-activated protein kinase activation. Endothelin- or thrombin-induced activation of mitogen-activated protein kinases was significantly inhibited by activation of cyclic AMP-dependent protein kinase by forskolin. Proliferation of airway smooth muscle cells, measured by incorporation of [3H]thymidine into DNA, was also greatly attenuated by forskolin.

Reactive Blending of Polyamide 6 and Polycarbonate

Abstract In this work structure and mechanical properties of blends of bisphenol-A polycarbonate with polyamide 6 with different molecular weights and different terminal groups are presented. The results show the important role played in the change of the morphology and in the improvements of the mechanical properties by the increasing content of NH2 terminal groups. These results are attributed to the reactions during melt mixing of such groups with polycarbonate.

Silver selective benzimidazol-2(1H)-one based, sulphur-containing podands

Podands based on benzimidazol-2(1H)-one, containing S and terminal NH2 groups, selectively transport and extract silver picrate over Mg2+, Ca2+, Sr2+, Ph2+ and Tl+ picrates.

Synthesis, spectroscopy and thermal properties of the nickel(II), palladium(II) and copper(II) complexes of bis(aminoalkyl)-oxamides (=LH2) including the crystal structure of Cu2L(NO3)2

The synthesis of the NiII, PdII and CuII complexes of N, N′-bis(aminoalkyl)oxamides (LH2) is described and structures are proposed on the basis of their physical and spectroscopic properties. With NiII and PdII only one complex is formed with general formula NiL or PdL, characterised by coordination through two deprotonated amide N-atoms and two terminal NH2 groups. With CuII it proved possible to obtain three structurally different compounds, depending on the pH, with general formulae Cu(LH2)X2, Cu2(L)X2 and CuL in which X=Cl, Br or NO3. The structure of [Cu2(C8O2N4H16](NO3)2 was solved by means ofx-ray diffraction; Mr=451.33, monoclinic, space group P21/n, a=9.503(4), b=7.614(1), c=10.407(3) A, β=98.43(3)°, V=744.3(7)A3, Z=2, Dx=1.202 g cm−3, μ=1.33 cm−1, γ(MoKα)=0.71073 A, F(000)=520, room temperature, R=0.043, wR=0.047 for 1080 observed [I>-3σ(I)] not systematically absent reflections out of 1423 measurements and 137 variables. The compound has a conformational chair/boat disorder with 82% in the more stable chair form.

Distinct epitopes on amiloride

Most Na(+)-selective transport proteins are inhibited by the drug amiloride. Studies using amiloride analogues suggest that specific regions of amiloride might participate in binding to receptors on these transport proteins. To determine whether certain domains of this drug are recognized as distinct epitopes, amiloride was coupled to albumin through either its C-5 NH2-group on the pyrazine ring or through a terminal NH2-group of the guanidino moiety, and antibodies were raised against these amiloride-albumin conjugates. Studies of antibody binding to amiloride analogues identified the 3,5-diaminopyrazinyl, the guanidinocarbonyl, and the C-6 halo moieties as distinct epitopes, although the antibodies required the presence of both the 3,5-diaminopyrazinyl as well as the guanidinocarbonyl moiety for binding.

Cloning of the cDNAs encoding the cellular retinaldehyde-binding protein from bovine and human retina and comparison of the protein structures.

A 1173-base pair cDNA encoding bovine cellular retinaldehyde-binding protein (CRALBP) was cloned from a bovine retinal cDNA expression library using as probes both anti-CRALBP polyclonal and monoclonal antibodies. The amino acid sequence deduced from the cDNA corresponds exactly to that determined by direct analysis of NH2-terminally acetylated bovine CRALBP (Crabb, J. W., Johnson, C. M., Carr, S. A., Armes, L. G., and Saari, J. C. (1988) J. Biol. Chem. 263, 18678-18687). Nick-translated bovine CRALBP cDNA probes were then used to clone from a human retinal cDNA library a 1317-base pair cDNA encoding human CRALBP. Bovine and human CRALBP are 92% identical in amino acid sequence and not related to any other known protein sequence. Both the bovine and human proteins contain 316 residues and have calculated molecular weights of 36,378 and 36,347, respectively, exclusive of the NH2-terminal blocking groups. The CRALBP cDNA clones should prove valuable as tools for studying the physiological role of the protein in vision and visual disorders.

Synthese der Spermidin‐Alkaloide (±)‐Inandenin‐10‐ol, Inandenin‐10‐on und (±)‐Oncinotin

Syntheses of the Spermidine Alkaloids (±)‐Inandenin‐10‐ol, Inandenin‐10‐one, and (±)‐OncinotineNew syntheses of the title compounds using two‐ring‐enlargement reactions are described. Starting from the aldehyde 1, the corresponding 4′‐aza derivative 15 could be obtained by reductive amination with the appropriate and protected spermidine derivative 14 (Scheme 4). Enlargement of the carbocyclic ring in 15 by five members gave, after further transformations, the hydroxylactam 18. Transamidation of 18, the second ring‐enlargement step, led to (±)‐inandenin‐10‐ol (7;22.9% overall yield) and, after oxidation, to inandenin‐10‐one (8; 22.5%, overall yield). (±)‐Oncinotine 6 was synthesized by two pathways (Scheme 6): protection of the terminal NH2 group by treatment with the Nefkens reagent and replacement of the OH group by Cl gave 24, which by thermal transamidation followed by direct ring closure led to the oncinotine derivative 26. The same intermediate could be obtained in higher yield via 28 by oxidation and protection of 18 followed by transamidation and reductive ring closure. Treatment of 26 with hydrazine finally gave (±)‐oncinotine 6 in 15.9% overall yield.

Specificity of binding of NH2-terminal residue of proteins to ubiquitin-protein ligase. Use of amino acid derivatives to characterize specific binding sites.

Previous studies have indicated that at least part of the selection of proteins for degradation takes place at a binding site on ubiquitin-protein ligase, to which the protein substrate is bound prior to ligation to ubiquitin. It was also shown that proteins with free NH2-terminal alpha-NH2 groups bind better to this site than proteins with blocked NH2 termini (Hershko, A., Heller, H., Eytan, E., and Reiss, Y. (1986) J. Biol. Chem. 261, 11992-11999). In the present study, we used simple derivatives of amino acids, such as methyl esters, hydroxamates, or dipeptides, to examine the question of whether the protein binding site of the ligase is able to distinguish between different NH2-terminal residues of proteins. Based on specific patterns of inhibition of the binding to ligase by these derivatives, three types of protein substrates could be distinguished. Type I substrates are proteins that have a basic NH2-terminal residue (such as ribonuclease and lysozyme); these are specifically inhibited by derivatives of the 3 basic amino acids (His, Arg, and Lys) with respect to degradation, ligation to ubiquitin, and binding to ligase. Type II substrates (such as beta-lactoglobulin or pepsinogen, that have a Leu residue at the NH2 terminus) are not affected by the above compounds, but are specifically inhibited by derivatives of bulky hydrophobic amino acids (Leu, Trp, Phe, and Tyr). In these cases, the amino acid derivatives apparently act as specific inhibitors of the binding of the NH2-terminal residue of proteins, as indicated by the following observations: (a) derivatives in which the alpha-NH2 group is blocked were inactive and (b) in dipeptides, the inhibitory amino acid residue had to be at the NH2-terminal position. An additional class (Type III) of substrates comprises proteins that have neither basic nor bulky hydrophobic NH2-terminal amino acid residues; the binding of these proteins is not inhibited by homologous amino acid derivatives that have NH2-terminal residues similar to that of the protein. It is concluded that Type I and Type II proteins bind to distinct and separate subsites of the ligase, specific for basic or bulky hydrophobic NH2-terminal residues, respectively. On the other hand, Type III proteins apparently predominantly interact with the ligase at regions of the protein molecule other than the NH2-terminal residue.

Specifically carboxymethylated hemoglobin as an analogue of carbamino hemoglobin. Solution and X-ray studies of carboxymethylated hemoglobin and X-ray studies of carbamino hemoglobin.

Hemoglobin can be specifically carboxymethylated at its NH2-terminal amino groups (i.e. HbNHCH2COO-) to form the derivatives alpha 2Cm beta 2, alpha 2 beta 2Cm, and alpha 2Cm beta 2Cm, where Cm represents carboxymethyl. Previous studies (DiDonato, A., Fantl, W. J., Acharya, A. S., and Manning, J. M. (1983) J. Biol. Chem. 258, 11890-11895) suggested that these derivatives could be used as stable analogues of the corresponding carbamino (Hb-NHCOO-) forms of hemoglobin, adducts that are generated reversibly in vivo when CO2 combines with alpha-amino groups. In this paper we present x-ray diffraction studies of both carbamino hemoglobin and carboxymethylated hemoglobin that verify this proposal and we use the carboxymethylated derivatives to study the functional consequences of placing a covalently bound carboxyl group at the NH2 terminus of each hemoglobin subunit. Our studies also provide additional information concerning the oxygen-linked binding of anions and protons to Val-1 alpha. Difference electron density analysis of deoxy alpha 2Cm beta 2Cm versus the unmodified deoxyhemoglobin tetramer (deoxy alpha 2 beta 2) shows that the covalently bound carboxyl moieties replace inorganic anions that are normally bound to the free NH2-terminal amino groups in crystals of native deoxyhemoglobin grown from solutions of concentrated (2.3 M) ammonium sulfate. In the case of the beta-subunits, the carboxymethyl group replaces an inorganic anion normally bound between the alpha-amino group of Val-1 beta, the epsilon-amino group of Lys-82 beta, and backbone NH groups at the NH2-terminal end of the F'-helix. In the case of the alpha-subunits, the carboxymethyl group replaces an anion that is normally bound between the alpha-amino group of Val-1 alpha and the beta-OH group of Ser-131 alpha. A corresponding difference electron map of carbamino deoxyhemoglobin in low-salt (50 mM KCl) crystals shows that CO2 bound in the form of carbamate occupies the same two anion binding sites. The alkaline Bohr effect of alpha 2Cm beta 2 is only marginally lower (approximately 7%) than that of alpha 2 beta 2. Previous studies (Kilmartin, J. V., 1977) have shown that about 30% of the alkaline Bohr effect is the result of an oxygen-linked change in the pK alpha of Val-1 alpha, and O'Donnell et al., 1979, found that this portion of the Bohr effect is the result of the oxygen-linked binding of chloride to Val-1 alpha.(ABSTRACT TRUNCATED AT 400 WORDS)

Regulation of surfactant secretion from isolated Type II pneumocytes by substance P

Substance P, an eleven amino acid neuropeptide, significantly inhibited release of [ 3H]phosphatidylcholine from pulmonary Type II epithelial cells in vitro. Basal release and release in response to the β-adrenergic agonist, terbutaline and 12- O-tetradecanoylphorbol 13-acetate (TPA) were significantly decreased in the presence of substance P. Inhibitory effects of substance P were noted following a 1 h exposure of primary cultures of Type II cells in vitro and persisted up to 3 h in the presence of the secretagogues, TPA and terbutaline. The IC 50 values for substance P inhibition of [ 3H]PC release were 10 μM for basal release, 40 μM for TPA-induced release and 50 μM for terbutaline-induced release. The related neuropeptide, physalaemin and the stable active analog of substance P, [pGlu 5, MePhe 8, MeGly 9]substance P [5–11], had no significant inhibitory effects on surfactant release whether in the presence or absence of TPA or terbutaline. These data support the hypothesis that NH2-terminal basic groups of substance P are necessary for inhibition of surfactant secretion from isolated Type II cells and support the concept that an inhibitory system contributes to mediation of surfactant secretion from Type II epithelial cells.

Separation of tryptic phosphopeptides of ribosomal origin by reversed-phase high-performance liquid chromatography

Phosphorylation sites for cyclic AMP-dependent kinase in ribosomal proteins and their synthetic analogues were converted to tryptic phosphopeptides and analysed by reversed-phase high-performance liquid chromatography (RP-HPLC) using gradients of acetonitrile in water and 0.1% trifluoroacetic acid. Tryptic variants differing by only NH 2-terminal basic amino acid redidues or phosphoryl groups were not always well resolved under these conditions. The different phospho forms could be resolved by RP-HPLC and thin-layer cellulose mapping was found to be the most effective strategy for the absolute purification of trytic phosphopeptides from crude tryptic digests.

Role of the alpha-amino group of protein in ubiquitin-mediated protein breakdown.

Previous studies suggest that the conjugation of ubiquitin to NH2 groups of proteins is required for protein breakdown. We now show that the selective modification of NH2-terminal alpha-NH2 groups of globin and lysozyme prevents their degradation by the ubiquitin proteolytic system from reticulocytes. The conjugation by ubiquitin of epsilon-NH2 groups of lysine residues, usually seen in multiples, was also inhibited in alpha-NH2-blocked proteins. Naturally occurring N alpha-acetylated proteins are not degraded by the ubiquitin system at a significant rate, while their nonacetylated counterparts from other species are good substrates. This suggests that one function of N alpha-acetylation of cellular proteins is to prevent their degradation by the ubiquitin system. alpha-NH2-blocked proteins can have their activity as substrates for degradation increased by incorporation of alpha-NH2 groups through the introduction of polyalanine side chains. Proteins in which most epsilon-NH2 groups are blocked but the alpha-NH2 group is free are degraded by the ubiquitin system, but at a reduced rate. It is therefore suggested that the exposure of a free NH2 terminus of proteins is required for degradation and probably initiates the formation of ubiquitin conjugates committed for degradation.

Action of peptidases in brain synaptic membranes on the NH 2-terminus of adrenocorticotropin using ACTH-(1–16)-NH 2 as a model substrate

The action of brain peptidases on NH2-terminal sequences of adrenocorticotropin was studied by incubation of ACTH-(1-16)-NH2 under different pH conditions. Profiles of metabolites and time course of product formation were obtained by HPLC analysis of the digests. Fragments of ACTH-(1-16)-NH2 were isolated and characterized by their amino acid composition and NH2-terminal groups. Both at pH 7.4 and pH 8.5 the following fragments were found: ACTH-(3-16)-NH2, ACTH-(4-16)-NH2, ACTH-(5-16)-NH2, and ACTH-(7-16)-NH2. At pH 7.4 the major products were ACTH-(4-16)-NH2 and ACTH-(7-16)-NH2, while the peptide ACTH-(3-16)-NH2 was the main metabolite at pH 8.5. The nature of identified peptides and the time course of their formation demonstrates that aminopeptidase activities predominate in the conversion of the NH2-terminus of adreno-corticotropin and related peptides by brain synaptic membranes.

Specific receptor sites for pyridoxal 5'-phosphate and pyridoxal 5'-deoxymethylenephosphonate at the alpha and beta NH2-terminal regions of hemoglobin.

hemoglobins have been prepared which are substituted with pyridoxal 5'-phosphate or pyridoxal 5'-deoxymethylenephosphonate at both NH2-terminal amino groups of either the alpha or the beta chains. 31P NMR titration curves of the 5' side chain show a substantial decrease in acid strength in hemoglobins pyridoxylated at the alpha chains and the reverse effect in the hemoglobins labeled in the beta chains. These changes can readily be explained by the interaction of the 5' side chains with surrounding residues which provide specific binding sites for the monoanion at the alpha chain termini and for the dianion in the case of the beta chains. Since the proportion of monoanion is at lest ten times greater for pyridoxal 5'-deoxymethylenephosphonate than for pyridoxal 5'-phosphate at neutral pH, the phosphonate reacts preferentially with the alpha chain NH2-terminal amino groups and the phosphate with the beta chain ones.

Labeling of hemoglobin with pyridoxal phosphate.

The reaction of pyridoxal 5'-phosphate (PLP) with deoxyhemoglobin is confined to 2 residues in the beta chains, i.e. the alpha-amino group of valine 1 and the epsilon-amino group of lysine 82, both of which are located in the polyphosphate binding site. The major product is a hemoglobin in which only the two NH2-terminal amino groups are substituted (symmetric diPLPHb). It is formed by subunit rearrangement of monoPLPHb which is the initial product of the pyridoxylation under anaerobic conditions. TetraPLPHb, with substitutions at lysine 82 and valine 1 of both beta chains is found as a minor component. It results from subunit exchange of asymmetric diPLPHb consisting of one unmodified alpha beta dimer and one which is pyridoxylated at both sites. Anaerobic electrophoresis and oxygenation curves show that this reaction is readily reversed by mixing the tetrasubstituted derivative with unmodified hemoglobin.

Metal-ion controlled reactions of 2,6-diacetylpyridine with 1,2-di-aminoethane and 2,6-diformylpyridine with o-phenylenediamine and the crystal and molecular structure of a pentagonal pyramidal cadmium(II) complex containing unidentate o-phenylenediamine

Reaction of 2,6-diacetylpyridine (dap) with an excess of 1,2-diaminoethane (en) in the presence of Ba[ClO4]2 in MeOH or EtOH at room temperature yields the complex [BaL2(ClO4)2]·H2O where the ligand L2 is the open-chain condensate of two molecules of dap with three molecules of en. The i.r. spectrum of the solid complex indicates that L2 exists in the di-imine form containing two imidazolidine rings derived from the nucleophilic addition of the two primary amine groups of the tetra-imine form across adjacent CN linkages. The use of higher reaction temperatures affords the complex [BaL1(ClO4)2] of the 18-membered macrocycle L1 derived from the [2 + 2] condensation of two molecules of dap with two molecules of en. The intermediacy of the co-ordinated open-chain ligand L2 in the L1 macrocycle formation is evidenced by the subsequent ring closure of [BaL2(ClO4)2]·H2O in the absence of added diketone; a transamination mechanism is proposed. Corresponding reactions of 2,6-diformylpyridine (dfp) with o-phenylenediamine (opd) in the presence of Ba2+ give complexes of the 18-membered macrocycle with the isolation of any intermediate. The single Ba2+ in [BaL2(ClO4)2]·H2O may be replaced by two Cu2+ with isomerization of L2 to the tetra-imine form, L3. The isomerization is reversed on treatment of Cu2L3(ClO4)4·2H2O with NCS–. The Ba2+in the macrocyclic complex [BaL1(ClO4)2] may also be replaced by two Cu2+ to yield Cu2L1(OH)(ClO4)3·H2O in which the two Cu2+ ions are intramolecularly bridged, and antiferromagnetically coupled, via the hydroxide ion. The use of Cd[ClO4]2·6H2O as template affords the complexes [CdL5(en)][ClO4]2 and [CdL6(opd)][ClO4]2 where L5 is the open-chain ligand derived from the condensation of one molecule of dap with two molecules of en, and L6 is the corresponding ligand derived from dfp and opd. Crystals of [CdL6(opd)][ClO4]2 are triclinic with a= 9.117(9), b= 10.797(8), c= 16.084(11)Å, α= 97.89(8), β= 81.43(9), γ= 70.70(10)°, Z= 2, and space group P. 2 560 Reflections above background were measured by a diffractometer and refined by full-matrix least-squares to R 0.065. The co-ordination geometry of the Cd atom is approximately pentagonal pyramidal, being bonded to the five nitrogen atoms (Cd–N 2.35–2.46 Å) of L6 in the pentagonal plane and to one NH2 group [Cd–N 2.341(9)Å] of the opd molecule in one axial position, the metal lying 0.42 Å above the equatorial N5 plane. The other NH2 group of opd is not co-ordinated. An incomplete X-ray analysis of [CdL5(en)][ClO4]2 indicates a seven-co-ordinate polymeric structure in which the en molecules bridge adjacent Cd2+ ions. Treatment of CdL6(opd)(ClO4)2·6H2O with Ni2+ in the presence of O2 yields the complex[Ni(L8)2][ClO4]2·H2O the properties of which suggest that the terminal NH2 groups of L6 have added across the adjacent CN bonds, this being followed by oxidative dehydrogenation to generate 2,6-di(2-benzimidazolyl) pyridine.

Tritium labeling of proteins to high specific radioactivity by reduction methylation.

The third component of human complement, hemoglobin A2, and IgG have been radiolabeled with tritium to specific activities of 179, 103, and 89 Ci/mmol, respectively, by reductive methylation. The labeling procedure is mild, requiring only brief exposure to formaldehyde (10 to 12 mM) and tritiated sodium borohydride (3 to 5 mM), and specific for the alpha amino groups of NH2-terminal residues and epsilon-amino groups of lysyl residues. The extent of modification for each protein ranged between 5 and 16% at available amino groups based on a stoichiometric reactivity of 2 mol of formaldehyde/mol of primary amine. Although the degree of substitution required to obtain these specific activities was high, no significant loss of antigenic or biological activities was apparent. Each labeled protein was 90 to 95% precipitable with antiserum elicited to the unreacted protein. The hemolytic activity of C3 was reduced by only 16%; the oxygen affinity of HbA2 was minimally decreased, and the phosphorylcholine binding affinity of an IgA myeloma protein (TEPC-15) was not significantly affected. Precise double antibody radioimmunoassays were developed with each tritiated protein capable of detecting less than 10(-8) g of antigen. Labeled protein samples have been stored for time periods up to 1 year without appreciable deterioration.

Calorimetric studies of carbon monoxide and inositol hexaphosphate binding to hemoglobin A.

Heats of CO and IHP binding to hemoglobin A have been determined under a variety of buffer and pH conditions. From these data heats of ion binding linked to hemoglobin oxygenation have been estimated. For IHP binding to deoxyhemoglobin the buffer-corrected enthalpies are surprisingly large, reaching -25 kcal/mol of IHP at pH 7.4. These values correspond to approximately -11 kcal/mol of proton absorbed upon IHP binding and may rise largely from the protonation of hitidine and NH2-terminal groups in the binding site (Arnone, A., and Perutz, M.F. (1974) Nature 249, 34-36). The decreased magnitude of delta HIHP observed at low pH parallels the decreased proton uptake at low pH. In 0.1 M chloride (pH 7.4) the reaction Hb(aq) + IHP leads to Hb x IHP(aq) has a standard free energy change (Edalji, R., Benesch, R.E., and Benesch, R. (1976) J. Biol. Chem. 251, 7720-7721) of -10 kcal and an enthalpy change of -25 kcal. Therefore, enthalpic forces provide the dominant driving force of this process. The origin of these large negative enthalpy changes is attributed to the exothermic protonation of protein basic groups induced by the proximity of phosphate negative charges. The importance of protonation in the binding of organic phosphates to hemoglobin may well extend to the specific binding of other phosphate substrates to enzyme reaction sites.

Die Kooperativität bei der CO2-Hb-Bindung: Messungen mit Hilfe der 13C-NMR-Spektrometrie / The Cooperativity of the CO2 Hb Binding: Measurement by 13C -NMR Spectrometry

The detailed structure of the CO2 Hb binding curve is of considerable physiological interest, because the carbamino derivates, which are formed by the reaction of CO2 with Hb, are respon­sible for the well known Haldane effect. Under physiological conditions CO2 binds to the four terminal NH2-groups of the protein chains of Hb and it is generally assumed, that there exists no cooperativity between these groups. To examine whether this assumption is correct or not, we have measured the CO2Hb binding curve over a wide PCO2-range (from 0 kPa to about 70 kPa) by using 13C-NMR spectrometry. Morrow and coworkers (1976) have shown that carbamino-Hb causes a specific NMR signal. The quantitative analysis of the NMR spectra were done according to the method of internal standard. By analyzing mixtures of known composition, we were able to show that this method is working. So we had to use the following equation: Z is the CO2 saturation of Hb, I is the intensity of the carbamino signal, and IHb is the intensity of the carbons of Hb, which are involved in the amide binding. (I/IHb)max is (I/IHb) at high PCO2, where Hb is completely saturated by CO2. For our experiments we used solutions of human desoxygenated Hb, which were equilibrated with a mixture of N2 and CO2. The CO2 contained 90% 13C-isotope. The pH was adjusted by titration to 7.2. The NMR measurements were mainly done with a Varian XL 100 NMR spec­trometer. The CO2 Hb binding curve, we have measured, is S-shaped and has an average Hill value (n̅) of 2.1. This result is quite striking with respect to the findings of other groups. A Hill value of 2.1 proves that CO2 like O2 binds to Hb in a cooperative manner. The discrepancy to the results of other groups may be explained by the fact that these authors measured the CO2 Hb binding curve only at comparatively low PCO2. In our opinion one can only get reliable n-values from the whole CO2 Hb binding curve.