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Oxidative Hydrolysis of Phosphorus Sulfides.

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Studies on the Active Centre of Ribonuclease
[7] D G. Herries, A . P. Mathias, and B. R . Rabin, Biochem. J .
85, 127 (1962).
E. A . Barnard, London (England)
[8] H. Wine1 and E. A. Barnard, Biochem.biophys. Res.Commun.
7,289, 295 (1962).
[9] W . A . Klee and F. M . Richards, J. biol. Chemistry 229, 489
(1957).
[lo] A. M . Crestfiekl, Fed. Proc. 22, 419 (1963).
[ l l ] E. A . Barnard and A. Ram(,/, Nature (London) 195, 243
(1962).
1121 H . Fraenkel-Conrat, Arch. Riochem. Biophysics 27, 109
(1 950).
[I31 E. A. Barnard and S. ShuN, unpublished results.
Virtually the only group in a n enzyme active centre that can
be fully identified and located, with reasonable confidence at
present, is the histidine residue 1 I9 in pancreatic ribonuclease.
It is difficult to prove that a group is a direct participant in
an active centre and is not only essential owing to some
other, protein structural, role. Direct participation appears
here to be substantiated by the convergence of evidence:
(1) Reaction of the enzyme with bromoacetic acid gives
specific, complete inactivation when one carboxymethyl
group is introduced [l]; this group is uniquely located at
histidine residue 119 [2].
(2) We have obtained the pure derivative by ion-exchange
chromatography; it closely resembles native ribonuclease in
,
physical properties, e . g . sedimentation constant ( S ~ O )spectrum, etc.
(3) The reaction of bromoacetate with the enzyme is much
faster than that with free histidine, and shows a n anomalous
dependence on p H (optimum p H 5.5).
(4) Reaction with iodoacetate [3] proceeds similarly; iodoacetamide does not react [4].
(5) The reaction requires the native enzyme; oxidized or
reduced ribonuclease does not react. It can occur to some
extent in urea [5] but not in all preparations [4]; this point
requires further investigation.
(6) Cytidine-2’- or -3’-phosphates are strong competitive inhibitors for ribonuclease and greatly protect it from attack by
bromoacetate.
(7) Photoxidation independently shows that a histidine is
essential for the enzymatic activity [6].
The dependence on p H of ribonuclease kinetics [7,8], as
well as of the halogenoacetate reaction, suggests that two
histidine residues are involved. That the second basic group
is not an -NH2 of low p K is shown by reaction of guanidinated [9] ribonuclease with bromoacetate, when the reaction proceeds with the same p H profile as that obtained with
native ribonuclease. Recently, direct evidence for participation of histidine residue 12 has been reported [lo].
1 19-Carboxymethyl-ribonucleasehas lost almost all binding
affinity for cytidine-2’-phosphate, as was confirmed by a
direct ultracentrifugal method [I I], suggesting histidine
residue 119 is at the substrate binding site. The kinetics of
each of the two steps of the enzyme-substrate reaction have
been determined on a series of substrates and support the
involvement of two histidines in binding the substrate.
Bromoacetic acid is thus the simplest case of a substrate-like
reagent, being bound as is the -P-O@ of the substrates,
to alkylate a nucleophilic component at the binding site.
In contrast to the behavior of ribonuclease, we find no comparable inactivation of lysozyme by bromoacetic acid
(0.18 M, p H 4 to 8.5). Lysozyme probably involves a histidine
residue in its active centre [12], but has uncharged substrates.
Other groups must also be concerned in the active centre of
ribonuclease. Recent studies [13] show the acylation of a
specific lysine residue, and allow its relationship to the
imidazole system to be explored.
[Lecture to the Basler Chemische Gesellschaft,
Base1 (Switzerland), April 25th, 19631
[VB 713/95 IE]
German version: Angew. Chem. 75,734 (1963).
[I] E. A . Barnard and W. D . Stein, J . molec. Biol. 1, 339 (1959).
[2] W. D. Stein and E. A. Barnard, J. molec. Biol. 1, 350 (1959).
[3] H.G. Gundlach, W. H . Stein, and S. Moore, J. biol. Chemistry
234, 1754 (1959).
[4] G . R . Stark, W. H. Stein, and S . Moore, J. biol. Chemistry
236, 436 (1961).
[5] E. A . Barnard and W. D. Stein, Biochim. biophysica Acta 37,
371 (1960).
[6] L. Weil and T. S. Seibles, Arch. Biochem. Biophysics 54, 368
(1955).
Angew. Chem. internat. Edit,
I
Vol. 2 (1963) / No. 9
Flash Photolysis and Fluorescence of Ammonia
W. Groth, Bonn (Germany)
A new flash arrangement for me in the vacuum ultraviolet
down to 1150 8, and based on the work of Bnyes, Becker,
Stufzl, and Welge was described. The reaction and discharge
chambers are separated by a scries of LiF windows. The
total energy of 1870 watt-sec is distributed over 12 parallel
spark gaps, which are flashed synchronously. The resolution
time can thus be decreased to . 2 psec. (A similar flash
arrangement for the quartz ultraviolet range with 5 parallel
spark gaps has a resolution timc of 3.7 psec. with a total
energy of 1440 watt-sec.).
During flash photolysis of ammonia, the N H radical was
detected in addition to the NH2 radical, at wave lengths of
< 1550 8, [(O,O)-and (1,l)-bands of the transition 3n + 3C-I.
The dependence of the N H concentration o n the delay time
of the analysis flash, on the NH3 pressure, and on the nature
of diluent gases was examined. The N H radical arises with
certainty as primary product of the photolysis by the reaction
NH3 + hv .+ NH -I- H2 or NH I 2 H.
During fluorescence analysis using the resonance wave lengths
of krypton (1165 8 , 1 2 3 5 8,) and xenon (1295 A, 1470 8,) only
the (0,O) bands of the 1II + 1A transition were detected, but
not the 311 + 3 F transition proposed by Neuimin and Terenin. There are two possible modes of formation of the N H
radicals in the 3% ground state that were detected by flash
photolysis: 1. direct transition into this state; 2. transition
to a singlet, with subsequent radiationless transition to the
3X- ground state. For a decision, experiments with greater
sensitivity of the equipment would be required. Upper limits
of the collision yields of reactions of the NH radicals can be
estimated.
During photolysis of NH3 with krypton and xenon lines, the
relative yield of hydrazine is the same in experiments, with
high gas flow rates, as those obtained with the Hg-line at
1849 A; the N H radicals must therefore react with NH3 to
giveN~H4.Thequantum yield of the NH3 decomposition with
the short wavelengths is greater by a factor of 1.3 to 1.6 than
that at 1849 8 . H2 in large excess has n o influence on the
quantum yield. On addition of CO, only formamide could
be detected as a further reaction product.
[GDCh-Ortsverband Mainz-Wiesbaden (Germany),
May 2nd, 19631
[VB 709192 IE]
German version: Angew. Chem. 75, 734 (1963)
Oxidative Hydrolysis of Phosphorus Sulfides
H. Falius, Braunschweig (Germany)
The work of Treadwell and Becli [l] and of Pernert and
Brown [2] o n the hydrolysis of phosphorus sulfides throws
no light on the constitution of the sulfides, owing to the
occurrence of redox and isomerization reactions. In the
[l] W.D. Treadwell and C. Beeli, Helv. chim. Acta 18,1161 (1935).
[2] J. C. Pernert and J. H . Brown, Chem. Engng. News 27,2143
(1949).
561
Presence of oxidizing agents, these reactions are retarded and
the P-P bonds in the sulfides, whose presence was demonstrated Some years ago by X-ray structural analysis, remain
unaltered. Since the P- P bonds in phosphorus oxyacids are
stable in alkaline solution and in order to avoid the presence
of foreign ions, the lower phosphorus sulfides P4S3, P4S5, and
P4S7 were hydrolysed i n solutions of various alkalinities with
hydrogen peroxide as oxidizing agent. The following oxyacids
of phosphorus were obtained (the percentage yields are based
on the initial quantity of phosphorus and are approximately
average values for all the hydrolyses):
P4S7: 10 % phosphite, 55 ”/, phosphate, 5 % pyrophosphate,
25 % hypophosphate, 5 triphosphate (V, IV, IV).
P4S5: 10 % phosphite, 40 ”/, phosphate, 10 % pyrophosphate,
25 % hypophosphate, 10 ”/, triphosphate (V, IV, IV), 5 % triphosphate (IV, 111, IV).
P4S3: 10 % phosphite, 35 % phosphate, 5 % pyrophosphate,
20 % hypophosphate, 15 ”/, triphosphate (V, IV, IV), 15 %
triphosphate (lV, 111. IV).
The qualitative and quantitative analyses were carried out
using paper chromatography with photometric measurements.
The stability of phosphite and hypophosphate in the reaction
solution was tested and confirmed. Pyrophosphate and triphosphate (V, IV, IV) must have been formed by a reaction
mechanism similar to that of the oxidative hydrolysis of thiophosphates to form pyrophosphates. This was investigated in
more detail. Starting from monothiophosphate 0 %, from
dithiophosphate 16 ”/,, and from tetrathiophosphate 12 ”/o
pyrophosphate (yield based on phosphorus) was formed.
[GDCh-Ortsverband Braunschweig (Germany),
May 13th, 19631
[VB 708/89 IE]
German version: Angew. Chem. 75,735 (1963).
strongly covalent and shortened. The P-l atom attempts to
pass on part of the electron charge to the oxygen bridge
atom 0 - 2 so as to enhance the polar character of the P(I)-0(2)
bond, which as a consequence is lengthened. The effect of the
cations should be such that a slight shortening of the
0(2)-P(2) bond is noticeable. This is shown especially clearly
in the interatomic distances of sodium triphosphate NasP30lo
(given in A).
1.49
r1.50
, 1.68
c)
1.61
y1.49
, 1.61
1.68
71.50
1.49
11.50
11.49
11.50
0
0
0
This effect of the cations in poly- and metaphosphates causes
the distance between the phosphorus atom and the oxygen
bridge atom to be about 1.62 A, while the remaining P - 0
distances, with an average of 1.49 A, are noticeably shorter.
In accordance with the character of the P-0 bond, the P-0-P
valence angle in the structures investigated so far deviates
only slightly from the mean value of 129O. The results of
crystallographic investigations are thus in harmony with the
theory of the chemical bond.
[Inorganic Chemistry Colloquium at the Universitlt
Gottingen (Germany), May 20th, 19631
tvB 714/90 l E ~
German version: Angew. Chem. 75, 735 (1963).
Recent Results in the Field of Homogeneohs
Catalysis of Gas Reactions
Z . G. Szabo, Szeged (Hungary)
Structural Chemistry of Crystalline Phosphates
F. Liehau, Wurzburg (Germany)
Considerations of the structures of inorganic phosphates can
begin either from [PO41 tetrahedra or from the coordination
polyhedra of the cations which can gradually be built up into
larger and larger units. In the former case, for example, one
proceeds from the discrete tetrahedra of monophosphates via
the double and triple tetrahedra of the di- and triphosphates
to the ring and chain form anions of meta- and polyphosphates. The possibility of linking [PO41 tetrahedra to form
layer and framework structures was also demonstrated. It is
not surprising that several phosphate structures are isotypic
with silicates.
In starting structural considerations from cation polyhedra,
one proceeds in a similar way, for example, from structures
with discrete [M(O,OH)(,] octahedra t o double and triple
octahedra and octahedron chains and layers. However,
whereas the [PO41 tetrahedra condense to share common
apexes, the cation polyhedra can also unite to share common
edges and faces.
The P - 0 bond in phosphates has some polar and some
covalent character. The four a-bonds, due to sp3 hybridization
are the most favorable energetically and give rise to the
tetrahedral arrangement of the oxygen atoms around the
The lecturer showed, by incans of his latest experimental
data, in particular by kinetic analysis of the thermal decomposition of propanal and acetaldehyde, that the theory
on the influencing of homogeneous chain reactions developed
by him and his co-workers can be confirmed exactly. Results
of experiments performed under conditions of utmost cleanliness in the presence and absence of nitric oxide were entirely
in conformity with the theoretical influencing factor and the
order of reaction was determined at 3/2 without deviation.
The values found for the thermodynamic variables were
entirely plausible. The agreement of theory and practice
shows that the influencing can be explained by assuming
radical stabilization, which is reversible and which has an
enthalpy of reaction of 15 kcal/mole. The theory permits
description of the mechanism of catalysis and inhibition of
homogeneous chain reactions from a unified standpoint and
it offers the possibility of investigating radical properties,
which would not be accessible to other methods. The relation
of the views developed by the lecturer to the theories of other
kinetic schools, such as those of Hinshelwood or of Eyring
were discussed
[GDCh-Ortsverband Ruhr, Mulheim/Ruhr (Germany),
May 29th, 19631
[VB 710/93 1El
German version: Angew. Chem. 75,798 (1963).
The Structure of the Intermediate in
Electrophilic Aromatic Substitutions
phosphorus atom. The free electron pairs of the oxygen
atoms are partially drawn into the d,z and the dXz-+ orbital
of the phosphorus to form two n-bond systems, which in
condensed phosphates are involved in the whole -0-P-O-P-Ochain. Since the cations (M) are less electronegative than the
phosphorus, the former act as electron donors and “push”
electrons from oxygen atom 0-linto the n-bond over towards
the phosphorus atom P-1. Hence the P(1)-0(1) bond is more
562
H. Zollinger, Zurich (Switzerland)
By suitable choice of reactants in electrophilic substitutions,
the relative velocities of the steps of electrophilic substitutions
can be varied widely. In suitable cases, the structure of the
intermediate in this type of substitution can thereby be
ascertained. Investigations of the nuclear magnetic resonance
of the intermediates obtaincd in the bromination and iodi-
Angew. Chem. i n t i w a t . Edit. / Vol. 2 (1963) I No. 9
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