THE EFFECT OF POLYHYDRIC AND MONOHYDRIC ALCOHOLS ON THE HEAT-INDUCED REVERSIBLE DENATURATION OF LYSOZYME AND RIBONUCLEASE

The reversible thermal denaturation of lysozyme and ribonuclease was investigated in aqueous solutions of mono- and polyvalent alcohols. The polyvalent alcohols show a stabilizing influence whereas the monovalent alcohols destabilize the native conformation. The concentration of the monovalent alcohol proportionally lowers the transition temperature Tm with concentration. The increments of Tm of lysozyme and of ribonuclease, excerted by a polyvalent alcohol, are of the same magnitude. This was also found for chymotrypsinogen A (1). It is reasoned that these stabilizing effects may find their origin in the intensification of the intra-hydrophobic interactions of the protein. The effects of the monovalent alcohols on the Tm of lysozyme as well as on the Tm of ribonuclease are enhanced with the increase of the hydrophobic character of the alcohol. These increments of Tm for lysozyme are slightly larger than those obtained for ribonuclease at 50–60°C, as could be expected from their amino acid compositions.     

DETECTION BY N.M.R. SPECTROSCOPY OF DEVIATIONS FROM RANDOM COIL BEHAVIOUR OF DENATURED PROTEINS

The acid denaturation of ribonuclease-A and lysozyme in 8 M urea has been studied by nuclear magnetic resonance (N.M.R.) spectroscopy and gel chromatography and compared with previous ultraviolet difference spectroscopic and viscometric studies. The N.M. R. results show that lysozyme is completely unfolded below pH 3 and virtually native at pH > 3.5 whereas Sigma ribonuclease-A is completely unfolded below pH 5 and about 60% native at pH 7. These results obtained at a protein concentration of 10% are consistent with the qualitative conclusions obtained by the other techniques in much more dilute solutions. The NMR method is an attractive procedure for rapid exploration of a range of solution conditions in order to find the best set of conditions for the separation of a mixture of proteins in the random coil form (I).     

CARBON-13 N.M.R. STUDY OF BLEOMYCIN-A2 PROTONATION

The acid-base titration of bleomycin-A₂ in D₂O solution at 35 ± 5° has been monitored by ¹³C n.m.r. spectroscopy at 67.89 MHz. The following pK^D_a values were obtained: 3.68 ± 0.05 (secondary amine), 5.29 ± 0.03 (imidazole), and 8.23 ± 0.19 (primary amine), where K^D_a is the dissociation constant in D₂O solution. The equilibrium isotope effects (pK^D_a -pK_a in H₂O) are: 0.70 ± 0.06 (secondary amine), 0.28 ± 0.04 (imidazole), and 0.85 ± 0.19 (primary amine). Titration of the imidazole group of Bleo-A₂ occurs at N^π, i.e. only N^π is protonated in basic solution. Significant protonation shifts are almost completely limited to carbons of the N-terminal tetrapeptide, suggesting that the C-terminal tripeptide extends into the solvent and interacts to a minimal extent with the rest of the molecule. Long range protonation shifts associated with titration of the imidazole and secondary amine groups indicate that protonation of one or both of these sites is probably accompanied by significant conformational changes. The observed protonation shifts generally fail to correlate with Zn(II) complexation shifts reported by Dabrowiak et al. (1973, Biochemistry 17, 4090) indicating that ligation sites cannot unambiguously be determined from these complexation shifts. The complexation shifts previously attributed to coordination of the imidazole and carbamoyl groups probably result from conformational changes.     

DISTANCE GEOMETRY ANALYSIS OF THE N.M.R. EVIDENCE ON THE SOLUTION CONFORMATION OF BLEOMYCIN

Conformational constraints derived from n.m.r. experiments, X-ray data and the known stereochemistry have been used to investigate by the distance geometry method the range of allowed solution conformations for Cu(II): P-3A (a biosynthetic precursor of bleomycin), Fe(II):bleomycin:carbon monoxide, and Zn(II): bleomycin. The experimental data have been found to be self-consistent and lead to the following observations. 1) Designation of the ligands and the dihedral angles available from vicinal coupling constants are not sufficient to define uniquely the geometry around the metal. 2) When only five bleomycin ligands are invoked (e.g. Cu(II):P-3A or Fe(II):bleomycin:carbon monoxide) there is considerable freedom in the allowed coordination scheme around the metal, but some regions of the molecule have well determined conformation. 3) Introduction of a sixth bleomycin ligand, as in Zn(II): bleomycin, considerably constrains the conformational freedom of the groups coordinated to the zinc. The utility of the distance geometry approach for analysis of data and design of experiments is discussed.     

N-ACYLSUCCINIMIDES AS ACYLATING AGENTS FOR PROTEINS: THE SELECTIVE ACYLATION OF LYSINE RESIDUES

The suitability of N-acylsuccinimides as reagents for the selective acylation of reactive side chains in proteins has been examined. A detailed study of the reaction of N-acetylsuccinimide with ribonuclease and bovine serum albumin showed that acetylation occurred in the pH range 3 to 8. Lysine side chains were acetylated selectively, with little or no modification of tyrosine side chains. There was no evidence for acetylation of serine, threonine or histidine side chains. The analogous N-propionyl, N-n-butyryl, N-i-butyryl and N-n-valerylsuccinimides also selectively acylate lysine side chains in proteins. This series of reagents, capable of changing the charge of lysine side chains and adding acyl groups of increasing hydrocarbon chain length, should prove a useful tool in studies of protein structure.     

1H N.M.R. STUDY OF THE CONFORMATION OF [Glu 4] OXYTOCIN AND ITS LANTHANIDE COMPLEXES IN AQUEOUS SOLUTION

At 400 MHz the ¹H chemical shifts, peptide NH-CαH coupling constants (J_Nα) and peptide hydrogen exchange rates of [Glu⁴] oxytocin in aqueous solution closely resemble those previously reported for oxytocin under comparable conditions, indicating that both the parent hormone and its analogue have similar conformations in this solvent. The hydrogen exchange data suggest a dynamic equilibrium between conformation(s) in which the peptide NH's of Asn⁵ Cys⁶ are internally hydrogen bonded and conformation(s) in which these hydrogens are bonded to the solvent. [Glu⁴]oxytocin forms 1:1 complexes with lanthanide metal ions. The diamagnetic La³⁺ complex exhibits values of J_Nα very similar to those of the metal free hormone analogue, suggesting that coordination of the metal is accompanied by minimal perturbation of the peptide backbone. Specific average proton-metal distances estimated from Gd³⁺ induced paramagnetic relaxation effects indicate that the metal is probably coordinated to the Glu⁴ carboxyl group and the sidechain carbonyl of Asn⁵. Limiting shifts induced by binding of paramagnetic Yb³⁺ are also reported.     

HYDROGEN ION TITRATION STUDY OF THE HISTIDINE RESIDUES OF HORSE MYOGLOBIN

The hydrogen ion titration curves of horse cyanometmyoglobin and metmyoglobin have been measured between pH 5.5 and 9.5. The most likely explanation of the titration results of cyanometmyoglobin is that 6 histidines are titratable with an intrinsic pK of 6.8 and an electrostatic interaction factor w of 0.150; in addition the results indicate that the total number of titratable carboxyl groups is 25, two more than calculated from the covalent structure of horse myoglobin. The titration behavior of metmyoglobin is explicable assuming the presence of one extra group as compared to cyanometmyoglobin with a pK near 9, which contributes to the maximum positive charge.     

LABELING OF AMINO ACIDS AND PEPTIDES WITH ISOTOPIC OXYGEN AS FOLLOWED BY 17O-N.M.R.*

¹⁷O was introduced into the respective α- and γ-COOH groups of Boc-Gly and Boc-Glu by saponification of the corresponding O-methyl esters with 1 N NaOH in H₂¹⁷O. Other ¹⁷O enriched Boc-amino acids were prepared by acid catalyzed exchange into the amino acid α-COOH group followed by t-butyloxycarbonylation with t-butyl S-4, 6-dimethylpyrimidin-2-ylthio carbonate. Final enrichment, by approximately three orders of magnitude over natural abundance, was 60–100% of the possible maximum. The synthesis of [¹⁷O]-Gly-Ala, [¹⁷O]-Gly-Leu and [¹⁷O]-Gly-Glu by DCC/HBT mediated coupling of Boc-Gly-[¹⁷'O]-α-COOH with amino acid-O-t-butyl esters followed by deprotection with HCl/EtOAc proceeded without undue loss of the isotope. Boc-[¹⁷O]-Pro-Leu-Gly-NH₂ was prepared by a similar procedure. [Tyr^2–17O]-, [Pro^7–17O]- and [Gly^4–17O]-oxytocin were synthesized using solid phase support. ¹⁷O-chemical shifts of synthetic intermediates and of the final products were as expected for each functional group. Linewidth data correlate with the molecular weights of the compounds prepared.     

FLUORESCENCE AND N.M.R. STUDIES OF THE BINDING OF CHOLINERGIC FLUORESCENT PROBES TO HORSE SERUM CHOLINESTERASE

The interaction of the cholinergic fluorescent probes, 1-(5-dimethyl-aminoaphthalene-1-sulfonamido) ethane-2-trimethylammonium perchlorate, 1-(5-dimethylaminonaphthalene-1-sulfonamido) pentane-5-trimethylammonium tartarate and 1-(5-dimethylaminonaphthalene-1-sulfonamido) decane-10- trimethylammonium tartarate with horse serum cholinesterase has been examined by fluorescence and n.m.r. methods. Fluorescence titrations show binding of the decane derivative to two sites on the protein whereas the lower homologs bind largely to one site. Active site inhibitors like curbamylcholine and decamethonium abolish binding of the decane derivative to the high affinity site. The inhibitors are largely without effect on the binding of the lower homologs. N.m.r. studies clearly establish immobilization of both ends of the molecule on binding in the case of the decane derivative, whereas in the lower homologs the dimethylamino group on the naphthalene ring is significantly more affected in the presence of enzyme. The probes are effective inhibitors of the enzyme with the decane derivative being two orders of magnitude more effective than its lower homologs. Based on the n.m.r., fluorescence and inhibition studies, a model for probe binding to the enzyme is advanced. It appears that the decane derivative binds with high affinity to the catalytic anionic site while the lower affinity site is assigned to a peripheral anionic site. The lower homologs probe only the peripheral site. A comparison of fluorescence, n.m.r. and inhibition studies with acetylcholinesterases from electric eel and bovine erythrocytes is presented.     

A COMPUTER-ASSISTED METHOD FOR DETERMINING THE NEAREST INTEGER RATIOS OF AMINO ACID RESIDUES IN PURIFIED PROTEINS

A routine method for finding the best set of integral amino acid residue numbers in a pure protein is described. The method starts with the best physical estimate of the molecular weight of the protein, and the amino acid composition derived from a number of amino acid analyses. The number of residues of each amino acid ± its standard error is calculated for the given molecular weight. Simple statistical screening procedures are used to eliminate unreliable amino acids. The remaining ones are used in a least-squares analysis developed from previous methods, to find the value of the molecular weight that gives the best fit to integral residue numbers. An estimate of the reliability of fit is also calculated. With the aid of a desktop computer the labour of finding the the best set of integers can be reduced to about 30 minutes' work. In addition an estimate of the confidence limits for each integer is provided. The use of the method is illustrated by reference to experimental results for bovine pancreatic ribonuclease, horse spleen apoferritin and E. coli citrate synthase.     

N.M.R. AND EQUILIBRIUM DIALYSIS STUDIES OF THE INTERACTION OF BOVINE NEUROPHYSIN-I WITH VASOPRESSIN AND SMALL PEPTIDES

The binding to bovine neurophysin of lysine-vasopressin and of lysine-vasopressin selectively deuterated at the protons ortho to the tyrosine hydroxyl was studied by proton n.m.r. and equilibrium dialysis. The principal object of these studies was to investigate reports that, at standard salt concentrations, neurophysin contained a second site specific for vasopressin. At pH 6, the effects of neurophysin-I on the line-width, longitudinal relaxation rate and nuclear Overhauser properties of the lysine-vasopressin tyrosine ring protons were interpretable in terms of a slow-exchange 1:1 interaction between lysine-vasopressin and neurophysin. Additionally, n.m.r. competition studies between lysine-vasopressin and L-phenylalanyl-L-tyrosinamide suggested 1:1 competition for a single binding site on neurophysin. No evidence pointing to a significant second lysine-vasopressin-binding site was obtained from the n.m.r. studies. The lack of a moderately strong second binding site for lysine-vasopressin at neutral pH was also indicated by equilibrium dialysis studies at relatively high free hormone concentrations. These studies demonstrated only a single thermodynamically significant site for either oxytocin or vasopressin and failed to confirm a reported effect of LiCl on the number of sites available to oxytocin. It is suggested that secondary sites for the hormones are probably markedly weaker and less specific than reported elsewhere.     

A STUDY OF THE CYSTINE RESIDUES IN BOMBYX MORI AND OTHER SILKS

Silks representative of the three subfamilies of Bombycidae have been studied and it has been found that they all contain cystine residues. The amount present varies, but irrespective of the country of origin is constant for a given species. In all cases the cystine content of the sericin was found to be at least twice that of the fibroin, which suggests that disulphide cross-linking does not contribute to the insolubility of the latter. The cystine content of Bombyx mori fibroin is 10.0 μ mole/g and this work has shown that at least one third of this cystine is present in the sequence Arg-Ala-Leu-Pro-Cys-Asn-Val-Cys Although such a small cystine-containing ring has not previously been reported as occurring in proteins, it is concluded that on stereochemical grounds the structure is sound.     

THE PREFERRED CONFORMATIONS OF PROTECTED HOMODI-TO HOMOHEPTAMETHIONINE PEPTIDES A 1H N.M.R. Study in Deuterochloroform Medium

Detailed analyses of the conformations of the homo-oligopeptide series, Boc-(L-Met)_n-OMe n = 2–7, in deuterochloroform have been carried out with proton n.m.r. and IR spectroscopy. Well-resolved high field n.m.r. spectra with assignments for the NH and α-CH resonances of these homo-methionine peptides are presented. Extensive n.m.r. concentration-dependent chemical shift studies are combined with IR results to delineate the involvement of the various methionine NH protons in intra- and/or intermolecular hydrogen bonding. N.m.r. chemical shift dependencies with temperature and solvent, DMSO-d₆, are used to explore the strength of the hydrogen bonds for the various oligopeptides. At low concentrations, where peptide aggregation is absent, the dipeptide is found to be disordered. The tetra- to heptapeptides possess intramolecular hydrogen bonded seven-membered rings at internal residues. The number of internal rings and the oligopeptide self-association increase with increasing peptide chainlength. At intermediate concentrations associations of peptide molecules with folded structures occur with initial association at the C-terminal region. At high concentrations, “in-register” associated extended β structures are formed.     

SYNTHESIS AND N.M.R. SPECTRAL ANALYSIS OF LEUCINE -dγ

A simple reaction scheme for the synthesis of leucine totally deuterated in the γ position is described. The n.m.r. spectral properties of this derivative are analyzed and its potential usefulness as a probe of leucine sidechain stereochemistry in peptides is discussed.     

小双孢菌新种研究Ⅰ.福建小双孢菌的鉴定

从福建省土壤中分离到五株有拮抗作用的小双孢菌,编号为80-11、80-14、80-71、80-74和80-182。这些菌株的气生菌丝体为灰色,基内菌丝体为红棕-紫褐色;在气生菌丝体的短孢子梗上产生呈纵对的孢子;在各种固体培养基上产生丰富的紫色结晶,在生长过程中,特别是气生菌丝体的生长必需有维生素B,在显微镜下观察菌体在生长过程中能产生分泌物附着于菌体表面。细胞壁化学组份Ⅲ型并含有少量甘氨酸,全细胞糖B型,含马杜拉糖。甲基萘醌为MK-9,MK-9(H_2)、MK-9(H_4)。通过分类研究,证明以典型菌株80-182为代表的几个菌株与小双孢菌属中的已知菌种有很大不同,是新种,命名为福建小双孢菌Microbispora fujianensissp·nov·1991。     

H N.M.R. STUDIES OF PROTECTED α-AMINOISOBUTYRIC ACID CONTAINING PEPTIDES

The benzylic methylene protons in a large number of benzyloxycarbonyl α-aminoisobutyric acid (Z-Aib) containing peptides, show chemical shift nonequivalence. The magnitude of the geminal nonequivalence is correlated with the involvement of the urethane carbonyl group, in an intramolecular hydrogen bond. Studies of the model compounds Z-Aib-Aib-Ala-NHMe, and Z-Aib-Aib-Aib-Pro-OMe clearly establish the presence of intramolecular hydrogen bonds, involving the urethane CO group. In both compounds marked anisochrony of the benzylic methylene protons is demonstrated. In Z-Aib-Aib-Pro-OMe, where a 4 → 1 hydrogen bonded β-turn is not possible, the benzylic -CH₂- protons appear as a singlet in CDCl₃ and have a very small chemical shift difference in (CD₃)₂SO. The observation of such nonequivalence is of value in establishing whether the amino terminal Aib-Pro β-turn is retained in large peptide fragments of alamethicin.     

APPLICATION OF THE ONE-DIMENSIONAL ISING MODEL TO THE SECONDARY STRUCTURES IN GLOBULAR PROTEINS.

An attempt has been made to predict the β-regions in 16 globular proteins by applying the one-dimensional Ising model theory of Lifson & Roig (8). The parameters for the theory have been derived from the statistical data on globular proteins given by Chou & Fasman (5). Comparison of our results with the data available from the X-ray crystallographic studies indicates a prediction accuracy which is comparable to those of several other methods, especially in view of the limitations in our method for considering the other secondary structures. It is pointed out that not considering the long-range interactions in our and other methods based on short-range interactions would make these methods incomplete and incapable of being uniformly applicable to all proteins.