bluuebunny/crossref_metadata_2025 huggingface dataset:
STUDIES ON TRYPSIN INHIBITORS
Authors: Tomatis Roberto , Guggi Augusto , Benassi Carlo A. , Salvadori Severo , Rocchi Raniero
Published: August 2010

The general strategy for the synthesis, by conventional procedures, of the entire sequence of porcine pancreatic secretory trypsin inhibitor II (Kazal) is discussed. The synthesis of two protected peptides corresponding to positions 2–10 and 1–10 of the proposed primary structure of the inhibitor is described. The heptapeptide free base threonyl‐S‐acetamidomethylcysteinylthreonylseryl‐γ‐tert‐butylglutamylvalylserine tert‐butyloxycarbonylhydrazide (sequence 4–10) was acylated, by the azide procedure, with either the dipeptide benzyloxycarbonyl‐γ‐tert‐butylglutamylalanine hydrazide (sequence 2–3) or the tripeptide Nω‐benzyloxycarbonyl‐Nω‐nitroarginylglutamylalanine hydrazide (sequence 1–3). The stereochemical homogeneity of the resulting peptides, benzyloxycarbonyl‐γ‐tert‐butylglutamylalanylthreonyl‐S‐acetamidomethyl‐cysteinylthreonylseryl‐γ‐tert‐butylglutamylvalylserine tert‐butyloxycarbonylhydrazide and Nω‐benzyloxycarbonyl‐Nω‐nitroarginylglutamylalanylthreonyl‐S‐acetamido‐methylcysteinylthreonylseryl‐γ‐tert‐butylglutamylvalylserine tert‐butyloxycarbonylhydrazide, was assessed, after partial deprotection with liquid hydrogen fluoride, by digestion with aminopeptidase M followed by quantitative amino acid analysis.
STUDIES ON TRYPSIN INHIBITORS
Authors: Guggi Augusto , Tomatis Roberto , Periotto Vittorio , Rocchi Raniero
Published: August 2010

Synthesis is described of the protected tetrapeptide corresponding to positions 11–14 of the primary structure of the porcine pancreatic secretory trypsin inhibitor II (Kazal), in the form of free acid as well as protected hydrazide. The tetrapeptide tert ‐ butyloxycarbonylglycyl‐S‐acetamidomethylcysteinylprolyl‐Nω‐trifluoroacetyl‐lysine was prepared by stepwise elongation from the C‐terminal Nω‐trifluoroacetyllysine using successively 1‐succinimidyl benzyloxycarbonylprolinate, p‐nitrophenyl N‐tert‐butyloxycarbonyl‐S‐acetamidomethylcysteinate and 1‐phenyl‐3‐methyl‐4‐(tert‐butyl‐oxycarbonylglycyl)‐oximinyl‐5‐(benzyloxycarbonylglycyl)‐imino‐2‐pyrazoline as acylating agents. Alternatively, the dipeptide benzyloxycarbonylprolyl‐Nω‐trifluoro‐acetyllysine was transformed into the corresponding tert‐butyloxycarbonylhydrazide which was reacted, after catalytic hydrogenolysis, with tritylglycyl‐S‐acetamido‐methylcysteine to give the tetrapeptide tritylglycyl‐S‐acetamidomethylcysteinylprolyl‐Nω‐trifluoroacetyllysine tert‐butyloxycarbonylhydrazide. The stereochemical homogeneity of the final products was assessed, after partial deprotection with aqueous 90% trifluoroacetic acid, by digestion with papain and aminopeptidase M, followed by quantitative amino acid analysis.
STUDIES ON TRYPSIN INHIBITORS
Authors: Tomatis Roberto , Guggi Augusto , Ferroni Roberto , Rocchi Raniero
Published: August 2010

The synthesis of the amino‐protected decapeptide tert‐butyloxycarbonylhydrazide corresponding to positions 15–24 of the amino acid sequence of porcine pancreatic secretory trypsin inhibitor II (Kazal inhibitor) is described. The tripeptide free base threonyl‐β‐tert‐butylaspartylglycine tert‐butyloxycarbonylhydrazide (sequence 22–24) was acylated with 1‐succinimidyl o‐nitrophenylsulfenylvalyl‐S‐acetamidomethy‐cysteinylglycinate (sequence 19–21). Removal of the amino protecting group from the resulting hexapeptide followed by acylation of the free base with either benzyloxy‐carbonylisoleucyl‐O‐tert‐butyltyrosylasparaginylproline or o‐nitrophenylsulfenyliso‐leucyl‐0‐tert‐butyltyrosylasparaginylproline, via the pyrazoline active ester method, yielded the decapeptide tert‐butyloxycarbonylhydrazide (sequence 15–24) in the form of Nω‐benzyloxycarbonyl or Nω‐o‐nitrophenylsulfenyl derivative. The stereochemical homogeneity of the two decapeptides was assessed, after partial deprotection with liquid hydrogen fluoride, or thioacetamide and aqueous 90% trifluoroacetic acid, by digestion with papain and aminopeptidase M followed by quantitative amino acid analysis.
KINETIC STUDIES OF THE ALKALINE MESENTERICOPEPTIDASE.
Authors: Rotanova Tatyana V. , Ivanova Anna G. , Antonov Vladimir K. , Rakadjieva Anastassia , Blagoev Blagoy
Published: August 2010

The inhibitory effect of alkylboronic acids H(CH2)nB(OH)2(n = 2–8) and Ph(CH2)nn‐ B(OH)2, (n = 0–4), on the alkaline mesentericopeptidase‐catalysed hydrolysis of synthetic substrates was studied. It was shown that alkylboronic acids act as bifunctional reversible inhibitors. The borate group interacts with an ionogenic group of the enzyme with a pKaof about 6.9‐7.0. The latter is probably the catalytically active imidazole of the active centre. The hydrocarbon part of the molecule also takes part in the formation of the enzyme‐inhibitor complex. The dependence of the degree of the enzyme‐inhibitor complex formation upon the length of the side‐chain of the inhibitor indicates the presence of two binding sites on the enzyme molecule.
DEAMIDATED ACTIVE INTERMEDIATES IN THE IRREVERSIBLE ACID DENATURATION OF RIBONUCLEASE‐A
Authors: Manjula B. N. , Acharya A. Seetharama , Vithayathil Paul J.
Published: August 2010

A study has been made on the changes in the enzymatic activity of Ribonuclease‐A**‐(RNase‐A) exposed to highly acidic (pH < 1) aqueous environment. Irreversible alterations of activity were observed when the protein was exposed to an acidic medium for a long period (20 to 60 h). Even prior to these changes in activity RNase‐A was found to form intermediates which had very nearly the same activity as the native protein.

The primary process in the acid denaturation of RNase‐A was observed to be deamidation of the protein leading to the formation of active chromatographically distinct derivatives. The initial product of deamidation, a monodeamidated derivative, has been isolated by chromatography on Amberlite XE‐64. This initial deamidation reaction proceeded with very high specificity. The subsequent deamidation reaction is comparatively slower, so that nearly 50% of the native protein could be converted to this derivative before any subsequent deamidation took place. This monodeamidated derivative has been designated RNase‐Aa1. The conversion of RNase‐A to RNase‐Aa1 was not accompanied by any changes in the primary structure other than the observed deamidation. Apart from the differences in chromatographic and electrophoretic mobilities, RNase‐Aa1 was found to have very nearly the same activity and physico‐chemical properties as the native enzyme. Significance of this specific and faster deamidation of RNase‐A in this denaturing medium as well as the biological significance of such deamidation reactions of proteins are discussed.
COVALENT COUPLING OF BILIRUBIN TO ALBUMIN
Authors: Jacobsen Christian
Published: August 2010

A method for covalent coupling of bilirubin to albumin is described. Human serum albumin‐bilirubin (1:1 complex) has been treated with water soluble carbodiimide in order to obtain covalent coupling of bilirubin to albumin. The reaction conditions have been varied with respect to pH, reaction time and concentration of reagent to obtain the optimal coupling. The prepared albumin‐bilirubin compounds were investigated by spectrophotometry, gel filtration and gel electrophoresis to ascertain the covalent nature of the bond and to characterize the products further. Gel electrophoresis and gel filtration showed that a monomer fraction could be prepared, and this fraction was a suitable material for further studies.
LASER RAMAN SPECTROSCOPY OF SNAKE VENOM NEUROTOXINS: CONFORMATION
Authors: Tu Anthony T. , Jo B. H. , Yu Nai‐Teng
Published: August 2010

Laser Raman spectra of neurotoxins of Pelamis platurus (yellow‐bellied sea snake) and Laticauda semifasciata (broad‐banded blue sea snake) were investigated. The amide I band appeared at 1672 cm‐1for both toxins, which presents an indication of anti‐parallel β structure. Since this agrees well with the result from the CD‐ORD studies of snake neurotoxin, it was concluded that snake neurotoxins mainly consist of β structure. The amide III band appeared at 1245 cm‐1for P. platurus toxin and 1248 cm‐1for L. semifasciata toxin.

The four disulfide bonds present in the toxin have a very similar geometry. After vigorous heat treatment, the backbone configuration of the toxin molecule basically remained the same although it was partially denatured. The major peak at 512 cm‐1was not altered by the heat treatment but a new shoulder appeared at 546 cm‐1. This suggests that a new type of S‐S stretching vibration (trans‐gauche‐trans) was produced as a result of heat treatment. However, the majority of the S‐S vibrations remained in the gauche‐gauche‐gauche orientation. A substantial change in the interactions between a tyrosine aromatic ring and neighboring residues was apparently the alteration caused by the heat treatment.
INTERPRETATION AND APPLICATIONS OF THERMAL DIFFERENCE SPECTRA OF PROTEINS
Authors: Nicola N. A. , Leach S. J.
Published: August 2010

The technique of thermal perturbation difference spectroscopy for examining chromophores in proteins has been set on a more acceptable theoretical and experimental foundation. (i) The origins of the thermal effect have been analysed to explain changes in spectral band width and wavelength for simple chromophores in various solvents. Comparison with theoretical curves shows that the effect of heating chromophore solutions is mainly spectral broadening coupled with an almost negligible blue shift. The apparently anomalous behaviour of tryptophan and tyrosine in aqueous solvents, where the main effect is a red shift on heating, is traced to hydrogen bonding with water. A model in which tyrosine acts simultaneously as H‐donor (Type I) and H‐acceptor (Type II) and the latter is the more temperature sensitive, is successful in explaining all available data for a variety of solute derivatives and solvents. (ii) The contribution of chromophores in the protein interior has been assessed using polyvinyl alcohol films of differing water content. These models simulate the rigidity and low thermal expansivity of the protein interior and confirm that buried chromophores contribute negligibly if at all to thermal difference spectra. (iii) Curve‐fitting procedures have been used for matching protein difference spectra over a wavelength range, to those for mixtures of models, rather than relying as hitherto on data measured at extrema.
THE PHOSPHOPYRIDOXYL PEPTIDE FROM THE MITOCHONDRIAL ASPARTATE AMINOTRANSFERASE OF BEEF KIDNEY
Authors: Bossa Francesco , Polidoro Giovanni , Barra Donatella , Liverzani Anna , Scandurra Roberto
Published: August 2010

The sequence around the coenzyme‐binding lysine of mitochondrial aspartate aminotransferase from beef kidney was determined. The holoenzyme was treated with NaB3H4 and digested with thermolysin; the labelled peptide was isolated and its sequence proved to be identical with sequences around the coenzyme site from the same isoenzyme of different organs and animals (pig heart and sheep liver). The sequences of the phosphopyridoxyl peptides of the mitochondrial aspartate aminotransferases appear to be closely related to the corresponding peptides from the cytoplasmic isoenzymes.
ON THE CATALYTIC ACTIVITY OF CHEMICALLY MODIFIED ENZYMES INVOLVING TWO OR MORE SUBSTRATES AND PRODUCTS
Authors: Südi J. , Havsteen B. H.
Published: August 2010

Catalytic activity of both native and chemically modified enzymes are numerically expressed in the literature with either kmax (= V/[ET]), or kE (= (V/[ET])/Km), if they refer to a one‐substrate one‐product reaction. Higher‐order catalytic constants of the k‘E type have not so far been calculated for enzyme reactions involving two or more substrates and products. a) It is shown here how such higher‐order k’E‐values can be calculated from easily obtainable experimental information; whatever the number of substrates and products, and even if binding of substrates and release of products follow a branched pathway, b) kE‐Values define the specific rate with which free substrates are converted by the enzyme into free products. Their dimensions (M‐nS‐1) are defined by the number of substrates(n). k‘E‐ Values refer to the same process as the catalytic constants generally used in physical organic chemistry, c) The required experimental information is mainly based on steady state parameters of the V’‐ and K‘m‐types, supplemented with equilibrium and fast kinetic studies of straightforward substrate binding reactions. d) It is shown that higher‐order k’E‐values always involve the “Haldane problem” of the chemical reaction step. On the other hand, k‘E‐values are independent of the rate of release of all but the first product in the overall reaction, e) The so‐called “Haldane relations” are shown to be determined by the way in which k’E‐values can be calculated from V‘‐ and K’m‐type experimental parameters, f) A number of theoretical considerations indicate that catalytic constants of the k‘E‐type are much more suitable for expressing the catalytic activity of chemically modified enzymes than maximal velocities are. This conclusion is demonstrated by a comparison of k’E‐ and k‘max‐values of some recently described modified forms of alcohol dehydrogenase.
ADRENOCORTICOTROPIN. 48. SYNTHESIS AND BIOLOGICAL ACTIVITY OF [15,16‐D‐LYSINE,17,18‐D‐ARGININE] ‐ADRENOCORTICOTROPIN‐(1–19) AND AN ALL‐D‐RETROPEPTIDE RELATED TO THE AMINO TERMINAL OCTADECAPEPTIDE OF ADRENOCORTICOTROPIN*
Authors: Blake James , Li Choh Hao
Published: August 2010

The peptide [15,16‐D‐lysine, 17,18‐D‐arginine]‐adrenocorticotropin‐(1–19) and an all‐D‐retropeptide related to the amino terminal octadecapeptide of adrenocorticotropin have been synthesized by the solid‐phase method. The nonadecapeptide was shown to possess 10–15% of the steroidogenic activity and 3% of the lipolytic activity of adrenocorticotropin‐(1–19). The all‐D‐retropeptide showed no activity and exhibited no inhibitory activity in steroidogenesis and lipolysis.
DES—Nα1—ACETYL—α—MELANOTROPIN:
Authors: Smeets Paul , Granger Michele , Nispen Johannes W. Van , Bloemendal Hans , Tesser Godefridus I.
Published: August 2010

Application of the 2‐methylsulfonylethyloxycarbonyl group for temporary amino protection enables the synthesis from one precursor of des‐Nα1‐acetyl‐α‐MSH, the two mono N‐acetylated forms (in positions I and II) and the diacetyl form of this tridecapeptide amide. The free tridecapeptide amide, although structurally unrelated to the normal substrate, was recognized by an enzyme occurring in calf eye‐lens tissue. The product of the enzymatic reaction was exclusively α‐MSH. Partial sequences derived from the N‐terminus were less rapidly acetylated or not at all, depending on their chain length. The enzyme, therefore, appears to direct its activity to free N‐terminal α‐amino groups of peptides exceeding a certain critical chain length. Acetylation of ‐amino functions did not occur.
BEE VENOM PEPTIDES XVIII. PEPTIDE‐M AND MCD‐PEPTIDE: ISOLATION AND CHARACTERIZATION
Authors: Jentsch Joachim , MüCke Hans‐Werner
Published: August 2010

A simple method is described for the purification of peptide‐M and MCD‐peptide from bee venom. The steps are Sephadex G‐50 gel filtration at pH 4.5 (Steps 1 and 2) and chromatography on silica gel (Steps 3 and 4). The peptides are homogeneous with respect to thin‐layer chromatography, electrophoresis at pH 2.0, and amino acid analysis. Peptide‐M contains 24 amino acid residues and has a calculated molecular weight of 2750.
STABILISATION OF ENZYME STRUCTURES BY INHIBITORS:
Authors: Das Manoj K. , Vithayathil Paul J. , Balaram P.
Published: August 2010

The acid denaturation of bovine pancreatic ribonuclease A in the presence of 0.2 M sodium dihydrogen phosphate has been studied by n.m.r. spectroscopy. Phenylalanine, tyrosine and methionine resonances serve as monitors of the unfolding process. It is shown that the inhibitor shifts the equilibrium towards the native structure at acid pH. Exchange broadening of the C‐2 resonances of the active site histidines, 12 and 119, occurs in the presence of phosphate, suggesting an equilibrium between native and unfolded structures. Stabilisation of the partially unfolded protein is observed at pH 1.5, as evidenced by the lack of the histidine resonance due to random coil protein. A scheme of the equilibria relating the various states of the protein is proposed.
STUDIES ON POLYPEPTIDES:
Authors: Voskuyl‐Holtkamp Ingrid , Schattenkerk Cecile
Published: August 2010

Syntheses of derivatives of the peptide sequences required for the synthesis of the N‐terminal 1–17 part of rat pancreatic ribonuclease are described. These protected peptides are:

OBut But OBut But But OBut

Boc‐Gly‐Glu‐Ser‐Arg‐OH (sequence 1–4); Nps‐Glu‐Ser‐Ser‐Ala‐Asp‐OH

Boc Boc

(sequence 5–9); Nps‐Lys‐Phe‐Lys‐Agr‐OH (sequence 10–13) and

OBut

Nps‐Gln‐His‐Met‐Asp‐OBut (sequence 14–17).
AGGREGATION, DISSOCIATION AND DENATURATION OF SESAME (Sesamum indicum L.) α‐GLOBULIN IN CETYL TRIMETHYL AMMONIUM BROMIDE SOLUTION*
Authors: Lakshmi T. S. , Nandi P. K.
Published: August 2010

The behaviour of the major protein of sesame seed (Sesamum indicum L.) α‐globulin has been studied in a cationic detergent, cetyl trimethyl ammonium bromide solution. Up to a critical detergent concentration the protein is precipitated from solution, above which redissolution of the protein is observed. Sedimentation velocity patterns indicate the presence of higher aggregates in the detergent concentration range 5 times 10‐5– 1 times 10‐3 M. These are considered to be the soluble precursors of the insoluble aggregates. Fluorescence measurements show that tryptophanyl groups of the protein which are in contact with the aqueous phase are perturbed by the detergent. The difference spectra of the protein in higher concentration of detergent indicate considerable red shift in the spectrum. Spectrophotometric titration of phenolic groups in 1 times 10‐2 M CTAB indicate that a conformational change in the protein has taken place.
CONFORMATION OF POLYPEPTIDE CHAINS CONTAINING BOTH L‐AND D‐RESIDUES:
Authors: Prasad B. V. Venkatram , Chandrasekaran R.
Published: August 2010

Polypeptides with alternating L‐ and D‐amino acid residues can take up stereochemically satisfactory coaxial double‐helical structures, both antiparallel and parallel, which are stabilized by systematic interchain NH O hydrogen bonds. Semiempirical energy calculations over allowed regions of conformational space have yielded the characteristics of these double‐helices.

There are four possible types of antiparallel double‐helices ‐ A3, A4, A5 and A6, with n, the number of LD peptide units per turn, around 2.8, 3.6, 4.5 and 5.5 respectively, while for the parallel double‐helices there are two types, P3 and P4, having similar helical parameters as in A3 and A4. The hydrogen‐bonding scheme restricts the pitch in all the models to the narrow range of 10.0 to 11.5 Å. All these helices have large central cores whose radii increase proportionately with n. In this respect, A3 and A4 are suitable models for the structure of gramicidin A.

In terms of their relative energies, antiparallel double‐helices are marginally more stable than those with parallel strands. Our results indicate that the energy differences amongst the members in the antiparallel family are not significant and thus provide an explanation for the polymorphism reported for poly(γ‐benzyl‐LD‐glutamate).
IMMUNOCHEMICAL INVESTIGATIONS OF HUMAN PITUITARY β‐LIPOTROPIN
Authors: Rao A. Jagannadha , Li Choh Hao
Published: August 2010

Antiserum to human β‐lipotropin has been raised in rabbits and characterized by gel double diffusion, quantitative precipitin, microcomplement fixation and biological neutralization tests. A sensitive radioimmunoassay for human β‐lipotropin has been developed. It was demonstrated that the antiserum shows specificity for this hormone. Human β‐melanotropin and human β‐endorphin showed only a minimal degree of cross‐reaction, while ovine β‐lipotropin showed a partial but significant degree of cross‐reactivity.
ACTIVE ESTERS IN THE FORMATION OF ESTER BONDS BETWEEN AMINO ACIDS AND POLYMERIC SUPPORTS
Authors: Bodanszky Miklos , Fagan Daniel T.
Published: August 2010

The imidazole catalyzed transesterification of active esters was used for the formation of the ester bond between the carboxyl group of protected amino acids and the hydroxyl group of a polymeric support applied in solid phase peptide synthesis. Anchoring of the C‐terminal residue to the hydroxymethyl polymer proceeded smoothly and provided a high degree of incorporation. No racemization was observed in the imidazole‐catalyzed alcoholysis. The procedure could be carried out with various active esters such as esters of o‐ and p‐nitrophenol, 2, 4, 5–trichlorophenol, pentachlorophenol and N‐hydroxysuccinimide.
HUMAN SOMATOTROPIN:
Authors: Noble Richard L. , Yamashiro Donald , Li Choh Hao
Published: August 2010

The amino terminal 54 residue peptide fragment of human somatotropin, [Cys(Cam)53]‐HGH‐(I‐54), has been synthesized by the solid‐phase method. The symmetrical anhydride and active ester coupling methods were used exclusively. The synthetic product was purified by gel filtration, isoelectric focusing, and partition chromatography. It was found to be homogeneous by six additional criteria.

In complement fixation experiments the synthetic product was immunologically active with antisera to HGH and [Cys(Cam)53]‐HGH‐(I‐134). Antiserum raised against the synthetic product was immunologically active in the homologous assay and with HGH, [Cys(Cam)53]‐HGH‐(I‐134), and [Cys(Cam)53]‐HGH‐(15–125). The synthetic fragment exhibited 53% of the activity of [Cys(Cam)53]‐HGH‐(I‐134) in the rat tibia assay.
A NOTE ON THE TWO‐DIMENSIONAL REPRESENTATION OF HYDROGEN BONDING SCHEME IN TRIOSE PHOSPHATE ISOMERASE*
Authors: Balasubramanian R.
Published: August 2010

When the backbone hydrogen bonding scheme of the protein triose phosphate isomerase is represented in the two‐dimensional map where the donor residue number (i) is taken along the x‐axis and the acceptor residue number (j) is taken along the y‐axis, the (i, j)‐map shows a characteristic pattern of (βα)8‐supersecondary structure.
PURIFICATION OF SEVERAL PROTEOLYTIC ENZYMES BY TOSYL‐ AND CARBOBENZOXY‐TRIETHYLENETETRAMINE‐SEPHAROSES
Authors: Fujiwara Kunio , Tsuru Daisuke
Published: August 2010

Tosyl‐triethylenetetramine‐Sepharose (Tos‐T*‐Sepharose) and carbobenzoxy‐triethylenetetramine‐Sepharose (Z‐T‐Sepharose) were found to be adsorbents utilizable in the purification of several microbial and animal proteases. The former Sepharose derivative adsorbed α‐chymotrypsin, trypsin, subtilisin, thermolysin and neutral subtilopeptidase at neutral pH range, and acid proteases such as pepsin and Rhizopus niveus protease at pH 3.5–6.5. α‐Chymotrypsin and trypsin were eluted with 0.1 N acetic acid and Rhizopus protease with 0.5 N acetic acid, thermolysin with 1 M guanidine ˙ HCI or 33% ethyleneglycol, whilst pepsin was recovered by elution with 2 M guanidine ˙ HCl at pH 3.5. The binding of neutral subtilopeptidase and subtilisin to this adsorbent was comparatively weak and both the enzymes were recovered by elution with 0.5 I M Nacl at neutral pH. On the other hand, Z‐T‐Sepharose was found to bind tightly to these proteolytic enzymes except neutral subtilopeptidase. Trypsin and α‐chymotrypsin were released from the adsorbent column with 1 M p‐toluenesulfonate, and subtilisin with 1 M guanidine ˙ HCl or 33% ethyleneglycol at neutral pH region. By these chromatographic procedures, the specific activities of these proteolytic enzymes increased effectively. Comparison of the binding abilities of acetyl‐, benzoyl‐, tosyl‐ and carbobenzoxy‐T‐Sepharoses to these enzymes suggests that hydrophobicity of tosyl and carbobenzoxy groups plays an important role in the enzyme‐adsorbent interaction.
SOLID PHASE SYNTHESIS AND SOME HORMONAL ACTIVITIES OF 8‐L‐HOMOARGININE‐VASOTOCIN AND 1‐DEAMINO‐8‐L‐HOMOARGININE‐VASOTOCIN
Authors: Larsson Lars‐Eric , Lindeberg E. Gunnar G. , Melin Per
Published: August 2010

8‐L‐Homoarginine‐vasotocin and its I‐deamino derivative were synthesized by the solid phase method. Both compounds possessed high activities in the uterus contraction and fowl depressor assays and showed higher pressor activities than the corresponding vasopressins. Homoarginine‐vasotocin, unexpectedly, was a more potent antidiuretic agent than its deamino analogue.
SPECIFIC CONFIGURATIONS OF HYDROGEN BONDING
Authors: Chen Cheng‐San , Parthasarathy R.
Published: August 2010

From a reexamination of the X‐ray studies of the crystal structures of 27 N‐acyl‐amino acids, peptides and their derivatives and 30 linear peptides, it is concluded that specific formation of short intermolecular hydrogen bonds (2.5 to 2.6 å) from the carboxyl OH to the N‐acyl oxygen is an important feature for N‐acyl‐amino acids. For N‐acyl‐N1‐amides, the formation of hydrogen bonds 2.7 to 2.9 å long between N(acyl)‐H … O(amide) is strongly preferred. The dihedral angle δ between the N‐acyl and carboxyl groups or adjacent amide groups shows a preference for values near 20° or 90° for N‐acylamino acids and 90° for N‐acyl‐N'‐amides.
AN AB INITIO INVESTIGATION OF MOLECULES WITH A DISULFIDE BOND: (HS)2, (CH3S)2 AND CYSTINE
Authors: Eslava L. A. , Putnam J. B. , Pedersen L.
Published: August 2010

ab initio Calculations at the Gaussian‐70 STO‐3G and 4‐31G basis levels have been carried out for (HS)2 and (CH3S)2. Cystine was investigated at the STO‐3G level. The STO‐3G energy minimized geometry agrees well with experiments for (HS)2 and (CH3S)2. The barriers to internal rotation are predicted to be (at the 4.31G level): (HS)2, cis 8.5 kcal, trans 3.03 kcal; (CH3S)2, cis 18.47 kcal, trans 6.04 kcal.
EMPIRICAL CALCULATIONS ON CYCLIC DIPEPTIDES
Authors: Genest M. , Ptak M.
Published: August 2010

Empirical conformational energy calculations were carried out for the dipeptides cyclo‐(L‐Ser‐L‐His) and cyclo‐(L‐Thr‐L‐His). Various DKP structures have been investigated to study side chain conformations through the DKP ring deformations, and prevalent conformers of monosubstituted and disubstituted dipeptides are discussed. The most stable conformations occur when both side chains are folded over a planar DKP ring, the imidazole ring being in the ε‐tautomeric form.

Theoretical results are consistent with those obtained from n.m.r. and crystallographic studies.
LOCATION OF DISULFIDE BRIDGES IN CARDIOTOXIN FROM THE VENOM OF FORMOSAN COBRA (NAJA NAJA ATRA)
Authors: Narita Kozo , Cheng King‐Lan , Chang Wen‐Chang , Lo Tung‐Bin
Published: August 2010

The location of the four disulfide bridges in cardiotoxin from the venom of the Formosan cobra, Naja naja atra, was determined by isolating cystine‐containing peptides from the enzymic and partial acid hydrolyzates of the intact toxin. The results indicate that the disulfide bridges are formed by half cystine residues 3–21, 14–38, 42–53, and 54–59. The amino acid sequence at the position 48–49 was revised to ‐Leu‐Val‐ instead of ‐Val‐Leu‐ as reported previously.
GRAPHICAL REFINEMENT OF THE AMINO ACID MOLAR RATIO AND OF THE MOLECULAR WEIGHTS OF PEPTIDES AND PROTEINS ON THE BASIS OF AMINO ACID ANALYSES*
Authors: MikeŠ Otakar
Published: August 2010

Corrected analysis results are handled graphically to find the most probable molar composition of polypeptides and proteins and to refine the molecular weights, even if only approximate molecular weights are available. The principle and properties of the graphical representation of idealized analyses are discussed first. Examples are given of the solution for a polypeptide consisting of 25 amino acids and for its not quite precise analysis and for a concrete protein ‐ extracellular alkaline protease from Aspergillus flavus. The general character of the method is discussed.
EFFECT OF SUCCINYLATION ON THE OLIGOMERIC STRUCTURE OF ARACHIN*
Authors: Shetty K. Jayarama , Rao M.S. Narasinga
Published: August 2010

Arachin succinylated to various levels has been studied by the techniques of sedimentation velocity, viscosity, absorption and fluorescence spectra, optical rotation and circular dichroism. Succinylation up to 60% leads to the dissociation of the parent 14S molecule to 9S and 4S fractions; above 60% succinylation, 4S fraction occurs predominantly. The other measured properties also show a gradual change up to 50% succinylation and a marked change above this level. The observed changes suggest that a new organised structure is formed before the 4S fraction is denatured.
THE SYNTHESIS AND OPIATE ACTIVITY OF HUMAN β‐ENDORPHIN ANALOGS
Authors: Blake James , Tseng Liang‐Fu , Chang Wen‐Chang , Li Choh Hao
Published: August 2010

Four analogs of human β‐endorphin have been synthesized and their opiate activities have been determined. One of the analogs, [27‐phenylalanine, 31‐glycine]‐βh‐endorphin, was shown to possess an analgesic potency greater than that of the natural peptide.
SYNTHESIS OF AN OPEN‐CHAIN ASYMMETRICAL CYSTINE PEPTIDE CORRESPONDING TO THE SEQUENCE A18–21 ‐B19–26 OF BOVINE INSULIN BY SOLID PHASE FRAGMENT CONDENSATION
Authors: Kullmann W. , Gutte B.
Published: August 2010

An insulin fragment containing residues A18–21 and B19–26 linked by the disulfide bond between residues A20 and B19 was synthesized. The sequence B21–26 was assembled on a solid support by the Merrifield technique. The protected fragments A18–21 and B19–20 were prepared by conventional methods. After forming the disulfide bridge through cleavage of the S‐thiocarbonate derivative of A18–21 by the thiol peptide B19–20, the resulting asymmetrical cystine peptide A18–21—B19–20 was coupled via the carboxyl group of residue B20 to the free NH2‐terminal amino group of the protected B21–26 resin. The product was deprotected, cleaved from the resin, and purified to give the homogeneous dodecapeptide A18–21‐B19–26.
CONFORMATION OF DISULFIDE BRIDGES IN PEPTIDES WITH PEPSIN PARTIAL SEQUENCES
Authors: Klis W. A. , Siemion I. Z.
Published: August 2010

On the basis of Raman spectra investigation of two model heterodetic cyclic peptides, containing partial sequences of pepsin fragments 45–50 and 206–210 of the chain, it was concluded that the disulfide bridge conformation in pepsin is determined not only by the size and conformation of the peptide loops created by disulfide bridges, but also by the peptide fragments located outside these loops.
DETERMINATION OF THE INTRAMOLECULAR TYROSINE‐TRYPTOPHAN DISTANCE IN A 7‐PEPTIDE RELATED TO THE C‐TERMINAL SEQUENCE OF CHOLECYSTOKININ
Authors: Schiller P.W. , Natarajan S. , Bodanszky Dr.M.
Published: August 2010

The solution conformation of a 7‐peptide with the C‐terminal sequence of cholecystokinin was investigated by evaluation of intramolecular resonance energy transfer between tyrosine (donor) in position 1 and tryptophan (acceptor) in position 4. From the relative enhancement of acceptor fluorescence a transfer efficiency of 0.70 ± 0.04 was determined. The use of this parameter in Förster's equation permitted the calculation of the average intramolecular tyrosine‐tryptophan separation, whereby the assumption of random donor‐acceptor orientation was made. The resulting average distance of 10.0 ±0.3 Å suggests some type of a folded conformation and excludes the existence of a fully extended chain in the N‐terminal part of the peptide. A comparison with tyrosine‐tryptophan distances observed in other biologically active polypeptides is made.
THE SYNTHESIS OF N‐BENZYLOXYCARBONYL‐L‐PROLYL‐DEHYDROPHENYLALANYL‐L‐HISTIDYL‐L‐LEUCINE
Authors: Konno S. , Stammer C.H.
Published: August 2010

The direct oxidation of a dipeptide azlactone (1) by DDQ affords an optically active unsaturated dipeptide azlactone (2). It was shown that the double bond had the Z‐configuration and that no racemization of the proline moiety occurred during the oxidation. The dehydrotripeptide, Z‐Pro‐ΔPhe‐Phe ˙ OH (9b) was prepared by direct coupling of azlactone (2) with phenylalanine tetramethyl‐guanidinium salt and was shown to be stable to chymotrypsin. The tetrapeptide, Z‐Pro‐ΔPhe‐His‐Leu ˙ OH (11), was prepared directly from the azlactone (2) and by saponification of the tetrapeptide ester (10) prepared by mixed anhydride coupling of Z‐Pro‐ΔPhe ˙ OH (7) with the dipeptide ester. The dehydropeptide (11) inhibits angiotensin I converting enzyme (IC50, 1 × 10‐4 M).
PROTEOLYTIC SUSCEPTIBILITY AND METHIONINE MODIFICATION OF MONODEAMIDATED RIBONUCLEASE A
Authors: Das Manoj K. , Vithayathil Paul J.
Published: August 2010

The susceptibility of a monodeamidated RNAaseA (RNAaseAa1) towards carboxypeptidaseA, α‐chymotrypsin and pepsin has been studied. Similar to RNAaseA, the C‐terminal of RNAaseAa1 is not available for carboxypeptidase A hydrolysis. The thermal stability of RNAaseAa1 as probed through chymotryptic digestion is found to be less than that of RNAaseA. Preliminary chromatographic analysis of the digested material, however, suggests that the nature of thermal transition might be the same in the two proteins. Pepsin inactivates RNAaseAa1 more slowly than does RNAaseA. Accordingly, less peptide bonds, almost half that of RNAaseA, are cleaved by pepsin in RNAaseAa1. The accumulation of RNAase‐P type intermediates is not evident during peptic digestion of RNAaseAa1. Reaction with O‐benzoquinone at low pH shows that methionines of the deamidated protein seem to have higher reactivities. These observations indicate a different structure for RNAaseAa1 at elevated temperature and low pH.
EMPIRICAL TORSIONAL POTENTIAL FUNCTIONS FROM PROTEIN STRUCTURE DATA Phi‐ and Psi‐Potentials for Non‐glycyl Amino Acid Residues
Authors: KOLASKAR A.S. , PRASHANTH D.
Published: August 2010

The torsional potential functions Vt(φ) and Vt(ψ) around single bonds N–Cα and Cα‐C, which can be used in conformational studies of oligopeptides, polypeptides and proteins, have been derived, using crystal structure data of 22 globular proteins, fitting the observed distribution in the (φ, ψ)‐plane with the value of Vtot(φ, ψ), using the Boltzmann distribution. The averaged torsional potential functions, obtained from various amino acid residues in l‐configuration, are Vt(φ) = – 1.0 cos (φ + 60°); Vt(ψ) = – 0.5 cos (ψ + 60°) – 1.0 cos (2ψ + 30°) – 0.5 cos (3ψ + 30°).

The dipeptide energy maps Vtot(φ, ψ) obtained using these functions, instead of the normally accepted torsional functions, were found to explain various observations, such as the absence of the left‐handed alpha helix and the C7 conformation, and the relatively high density of points near the line ψ = 0°. These functions, derived from observational data on protein structures, will, it is hoped, explain various previously unexplained facts in polypeptide conformation.
N.M.R. OF ‘ORIENTED’ MONOSUBSTITUTED AMIDES—CIS AND TRANS N‐METHYL FORMAMIDE
Authors: RAMAPRASAD S. , KUNWAR A.C. , KHETRAPAL C.L.
Published: August 2010

The n.m.r. spectrum of N‐methyl formamide oriented in a lyotropic liquid crystal solvent shows the presence of the cis as well as the trans forms. Analysis of the spectra due to both the species has been carried out. Possible non‐planar distortions at the nitrogen atom for the trans species have been computed using the derived dipolar couplings and the available structural data.
PROTAMINES
Authors: MARCHIORI FERNANDO , BORIN GIANFRANCO , FILIPPI BRUNO , MORETTO VITTORIO , BONORA GIAN MARIA , TONIOLO CLAUDIO
Published: August 2010

The synthesis of peptides containing blocks of arginyl residues is proposed through amidination of the corresponding ornithyl analogs. In order to test this strategy the ornithyl analog of the C‐terminal sequence 52–65 of galline was synthesized by the conventional method. The amidination reaction, performed on fragments of different length and ornithyl‐residue content, quantitatively converts ornithines into arginines. The strategy proposed may represent a powerful tool for the synthesis of protamines and other basic proteins.
SIDE REACTIONS IN PEPTIDE SYNTHESIS
Authors: MARTINEZ JEAN , TOLLE JOHN C. , BODANSZKY MIKLOS
Published: August 2010

Acylation of the hydroxyl groups in the side chains of serine, threonine or tyrosine occurs in coupling with active esters. This side reaction, which is quite pronounced in histidine‐containing peptides, can be prevented with additives. From a series of compounds tested, 2,4‐dinitrophenol and pentachlorophenol were the most effective.
IDENTIFICATION AND OBSERVATION OF ALKYL PROTON RESONANCES OF THE AMINO‐TERMINAL RESIDUES OF BOVINE NEUROPHYSINS
Authors: LORD SUSAN T. , BRESLOW ESTHER
Published: August 2010

Analysis of the 220 MHz proton magnetic resonance spectra of bovine neurophysins‐I and ‐II and of the effects of pH and succinylation on these spectra has allowed identification of the ‐CH3proton resonances of the amino‐terminal alanine of both proteins and of the ‐CH3resonance of methionine‐2 of neurophysin‐II. The alanine ‐CH3resonance of neurophysin‐I is a sharp doublet at all pH values between 1 and 10.5 indicating relatively few restrictions on its mobility. By contrast, the ‐CH3resonances of the amino‐terminal alanine and methionine‐2 of neurophysin‐II undergo pH‐dependent changes in broadening compatible with the formation of an intramolecular salt‐bridge at neutral pH between the protonated α‐amino and an unprotonated side chain carboxyl. The results suggest that differences in the properties of the two proteins are partially mediated by conformational differences involving their amino‐terminal sequences. The potential usefulness of the amino‐terminal resonances as n.m.r. ‘reporter’ signals is additionally demonstrated by studies of the effects of spin labels on the neurophysin‐I amino‐terminal alanine resonance; these studies place the amino‐terminus of neurophysin‐I approximately 14 Å from residue 3 of peptides bound to the strong neurophysin hormone‐binding site.
SYNTHESIS, CHARACTERIZATION AND FLUORESCENCE STUDIES ON N‐α‐DANSYLALANYLLYSYL TRYPSINO (HIS‐46)‐METHANE
Authors: PENNY GLENN S. , DYCKES DOUGLAS F. , BURLEIGH B. DANIEL
Published: August 2010

The 1‐dimethylamino‐5‐naphthalene sulfonyl (dansyl) derivative of alanyllsyl chloromethane was synthesized and employed to introduce the fluorescent dansyl moiety specifically into the active site of trypsin via affinity labelling. The potential of dansylalanyllysyl chloromethane lies in its high degree of selectivity and markedly faster rate of enzyme inactivation when compared to previously synthesized, single residue affinity label chromophores. This permits the practical utilization of stoichiometric amounts of the inhibitor to achieve 100% inactivation of trypsin, even at high dilutions.

The transfer of energy between the four tryptophan residues of trypsin and the bound dansyl group has been investigated in the fluorescent inhibitor‐enzyme conjugate. From transfer efficiency measurements mean distances of 19.0 Å and 19.3 Å between the point of attachment of the dansyl group and the four tryptophan residues of trypsin have been calculated. These compare well with the mean value of 18.8 Å derived from calculations based on crystallo‐graphic model studies.
COMPARISON OF BUNGARUS CAERULEUS VENOM WITH THE VENOM FROM WHICH A PUTATIVE CHOLINERGIC IONOPHORE MARKER WAS ISOLATED
Authors: NOBLE RICHARD L. , COOPER MARK J. , HESS GEORGE P.
Published: August 2010

Comparisons are described between Bungarus caeruleus venom and the actual venom from which a putative marker for the cholinergic ionophore, called ceruleotoxin, was isolated. The venoms are shown to be different by two procedures for ion exchange chromatography and by isoelectric focusing on polyacrylamide gel. The activities of the purified “ceruleotoxin” as an inhibitor of acetylcholine receptor‐mediated ion flux and as a phospholipase have been reported (Bon & Changeux, 1977b). The results reported herein suggest that this toxin is from an unknown origin.
ISOLATION AND CHARACTERIZATION OF FIN WHALE PROLACTIN*
Authors: KAWAUCHI HIROSHI , TUBOKAWA MAKOTO
Published: August 2010

A highly purified prolactin preparation has been obtained from fin whale pituitaries by extraction with acid acetone, salt precipitation, isoelectric fractionation, and exclusion chromatography on Sephadex G‐100 and ion‐exchange chromatography on DEAE‐cellulose. Fin whale prolactin was isolated in a yield of 250 mg/kg wet weight tissue. It was found to have a molecular weight (SDS disc gel electrophoresis) of 23,600 daltons and an α‐helix content (circular dichroism) of 50%. The amino acid composition and circular dichroism spectra were very similar to those of porcine prolactin. The partial amino acid sequence has been determined by the method of fluorescein‐isothiocyanate. Fin whale prolactin was found to be 80% as potent as ovine prolactin with regard to pigeon crop‐sac assay.
pH DEPENDENCE OF THE CIRCULAR DICHROIC BANDS OF PHENYLMETHANESULFONYL‐MESENTERICOPEPTIDASE
Authors: SHOPOVA MARIA , GENOV NICOLAY
Published: August 2010

The effect of pH on the circular dichroism spectra of phenylmethanesulfonyl‐mesentericopeptidase (peptidyl peptide hydrolase, EC 3.4.21) was studied. The ellipticity of the bands below 250 nm, which reflects the backbone conformation of the protein molecule, remains almost unchanged in the pH range 6.2–10.4. However, below pH 6.2 and above pH 10.4 a conformational transition occurs. The pH‐dependent changes above 250 nm were also studied. The titration of the CD band at 296 nm reflects the ionization of the “exposed” tyrosines, which phenolic groups are fully accessible to the solvent. An apparent pK of 9.9 is calculated from the titration curve. It is concluded that ionization of the tyrosyl residues with normal pK's is complete before conformational changes in the protein molecule occur.
ISOLATION AND PHYSICAL CHARACTERIZATION OF BOVINE LENS CRYSTALLINS
Authors: CHIOU SHYH‐HORNG , AZARI P. , HIMMEL MICHAEL E. , SQUIRE PHIL G.
Published: August 2010

The soluble proteins from bovine lens homogenate were separated on Sepharose CL‐6B (2 times 200 cm) in 0.05 m tris‐NaHSO3 pH 8.2 buffer containing 20 mM EDTA. Five sharp and defined fractions (HMα, α, βH, βL, γ) were obtained. Each crystallin fraction was further purified by rechromatography on the same column. Each protein fraction was pure as judged by ultracentrifugation and SDS‐gel electrophoresis. The molecular weights of the five fractions were 3.04 times 106, 5.83 times 105, 1.58 times 105, 4.59 times 104, 2.14 times 104 as determined from sedimentation coefficient and intrinsic viscosity data by Scheraga‐Mandelkern equation, which was in close agreement with that obtained by gel filtration. The polypeptide composition of crystallins as determined by SDS‐gel electrophoresis revealed one band for high molecular weight α (HMα) and α, three for βH, two for βL and one for γ.

The gross CD patterns of crystallins were about the same in the peptide region (200 nm ˜ 250 nm) with a minimum centered at about 217 nm, indicative of α β‐sheet structure in all crystallins. The [0] values at 217 nm ranged from –1700 to –3700 degrees cm2 per decimole. The CD spectra of these crystallins in the aromatic region (250 nm ˜ 300 nm) were different, reflecting the different contributions of aromatic amino acids to the tertiary structure of crystallins.
THERMAL ACTIVATION OF PEROXIDASE FROM TOBACCO LEAF MESOPHYLL CELL WALLS
Authors: CHOY YUEN‐MIN , WONG YUM‐SHING , LAU KIT‐MAN , HYUNG KUNG
Published: August 2010

Cell wall‐bound peroxidase isolated from tobacco leaf mesophll cell walls was found to consist of two isoenzymes (P1 and P2). Each exists in the form of a single subunit with the same molecular weight (35 000) as determined by Sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis. Both isoenzymes exhibited maximum activities at 70°. P1 increased to 265% and P2 to 140% of the activities assayed at 27°; below this temperature the two isoenzymes had the same specific activity. On hydrolysis, P1 showed a carbohydrate content of 26.22% when the monosaccharides were analyzed by gas liquid chromatography; P2 gave 21.45%.
METHIONINE ENKEPHALIN AND ISOSTERIC ANALOGUES I. Synthesis on a Phenolic Resin Support†
Authors: HUDSON DEREK , KENNER GEORGE W. , SHARPE ROBERT , SZELKE MICHAEL
Published: August 2010

An efficient synthesis of methionine enkephalin using a phenolic resin support is described. Analogues modified at their C‐termini, such as peptide acids, amides, methyl esters and compounds formed by their reduction, were prepared conveniently from common peptide phenyl ester resins. The resin was used in the synthesis of complex isosterically modified analogues designed to investigate the role the peptide backbone plays in receptor interaction. Free hexapeptide phenyl ester resins underwent intramolecular aminolysis liberating the corresponding cyclic peptides.
TRYPSIN AND CHYMOTRYPSIN INHIBITOR FROM CHICK PEAS Selective Chemical Modifications of the Inhibitor and Isolation of Two Isoinhibitors
Authors: SMIRNOFF PATRICIA , KHALEF SHULAMITH , BIRK YEHUDITH , APPLEBAUM SHALOM W.
Published: August 2010

The trypsin and chymotrypsin inhibitor from chick peas (CI) is stable in HCl 0.001 m – 0.01 m and in KOH 0.01 m – 0.05 m even after 24 h. Increased KOH concentrations decrease considerably the inhibitory activity already after 1 h. Maleylation and succinylation of the inhibitor resulted in almost full loss of its trypsin‐inhibitory activity but had no effect on the chymotrypsin‐inhibitory activity. A series of modifications directed towards tyrosyl residues showed that iodination influenced only the chymotrypsin‐inhibitory activity; however, nitration and arsanilation affected not only the chymotrypsin‐inhibitory activity but also the trypsin‐inhibitory activity. Treatment of the inhibitor with CNBr and chloramine T resulted only in a decrease in the chymotrypsin‐inhibitory activity indicating that the only methionine is involved in the chymotrypsin‐inhibitory activity. When CI‐fragment A, previously treated with trypsin at pH 3.75, was further treated with carboxypeptidase B, a release of three lysyl residues per mole protein was found. CI was separated by equilibrium chromatography on SP‐Sephadex column into two isoinhibitors, CII and CIII, respectively. Both inhibited trypsin and chymotrypsin with the same specific activity as CI. They differed from each other only in a glutamyl, aspartyl, glycyl and alanyl residue.
SYNTHESIS OF A SERIES OF RESIDUE 1 (PYROGLUTAMIC ACID) ANALOGS OF THYROTROPHIN RELEASING HORMONE
Authors: BAUCE LORENZO G. , GOREN H. JOSEPH
Published: August 2010

This report describes the synthesis of 23 analogs of thyrotrophin releasing hormone,l‐pyroglutamic acid‐l‐histidyl‐l‐prolineamide, where only the pyroglutamic acid moiety is modified. Twelve of the analogs contain different heterocyclic rings or are derivatized pyrrolidone rings. The syntheses of these pyroglutamic acid analogs are also described. Peptide bond formation was generally achieved by catalyzing carbodiimide coupling with 1‐hydroxybenzotriazole. The histidine side chain was protected with the 2,4‐dinitrophenyl group during di‐ and tripeptide synthesis and was removed with benzenethiol. Final purification of tripeptides involved passage of synthetic material over a Dowex 1 – X4 (bicarbonate) column. The route for synthesis of thyrotrophin releasing hormone and its analogs is highly efficient, since the native hormone may be obtained with 90% efficiency.
PREPARATION AND SOME PROPERTIES OF A DIMERIC FORM (S‐S) OF HORSE MUSCLE ACYLPHOSPHATASE
Authors: STEFANI MASSIMO , BERTI ANDREA , CAMICI GUIDO , MANAO GIAMPAOLO , CAPPUGI GIANNI , RAMPONI GIAMPIETRO
Published: August 2010

The use of sodium selenite as a catalyst in the presence of oxygen was a suitable technique to obtain in good yield an interchain S‐S dimeric form of horse muscle acylphosphatase. The dimer so obtained possesses kinetic properties very similar to those of the native enzyme. On the other hand the dimer has shown a generally lower stability in respect of the thermal inactivation, particularly in the acidic environment, to the lyophilization and to the proteolytic attack. As regards the 8 m urea inactivation, the dimer is not able to completely regain its activity by dilution, showing a behaviour quite different from that of the native enzyme.