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Cleavage of Proteins without Enzymes.

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of a velocity constant which is independent of concentration
(k, in Table 1, given in the form of its negative logarithm,
pk). This velocity constant is a measure for the acidity of the
Table I . Acidity Values
pk(25 “C)mpK,(25 “C)
Chlorosulphonicacid
[RzOHl+SbClsPerchloric acid
Sulphuric acid monomethyl ester
p-Toluenesulphonicacid
[R20H...OR21+SbClsTriRuoroacetic acid
Trichloroacetic acid
Picric acid
Monochloracetic acid
-11.27
-10.50
-10.08
- 7.76
- 5.48
- 4.70
- 2.51
- 1.40
+ 0.52
+ 1.54
acid concerned. Since the ranges of sensitivity of the three
diazo-compounds overlap each other and therefore the
values of k obtained with them may be related to one another,
it was possible to establish a continuous series of acidities
over almost 13 pk-units (up to chlorosulphonic acid). On
comparison with the “normal” series of acidities obtained
from acid-base equilibria, it appeared that the usual pKa units
could be equated with the pk-units determined by us, at least
to a first approximation, and that the pk values obtained by
the use of (111) at 25°C are (fortuitously) almost equal
numerically to the usual pKa values in water. From the
figures compiled in Table 1, the following observations can be
made: 1. R20H+ions not stabilised by hydrogen bonds have
pk values of -10.50 and therefore belong to the strongest
acids. 2. The decrease in acidity due to hydrogen bond
formation amounts to about 6 pk-units per hydrogen bond,
which was to be expected considering that the energy of
formation for the hydrogen bonds of strongly acidic substances is about 8 kcal/mole. 3. Strong acids (from p-toluenesulphonic acid onwards) which do not tend to form double
molecules are essentially stronger in ethylene chloride than
in aqueous solution, due to hydration. 4. The extraordinarily
high acidity of the R20H+ ion clearly explains the unusually
large acylation capacity of its esters, the trialkyloxonium ions.
[GDCh Ortsverband Freiburg - Siidbaden, Freiburg/Brsg.
(Germany) July 20th, 19621.
[VB 628/49 IE]
-
sequence is known with certainty. Such certainty is difficult
to obtain with the conventional methods of sequence determination which have been elaborated into an exacting and
complex “book-keeping” or “accounting” procedure in
which overlapping peptide sequence must properly add up
to a correct total sequence. The “auditing” of such sequences
requires non-enzymatic selective cleaving agents which
should preferably split peptide bonds that are not attacked
by enzymes. Such a cleaving agent is, for example, cyanogen
bromide, which selectively and almost quantitatively cleaves
the four peptide bonds following the four methionine residues
in ribonuclease at room temperature and acidic pH [3]. These
cleavages are free of side reactions and lead to three fragments: a core, a chemical tail peptide containing the amino
acids 1-13 and free homoserine. If the “S-peptide” containing the amino acids 1-20 of ribonuclease is subjected to
cleavage by cyanogen bromide, the separation of the products
on columns of cyclodextran (Sephadex) led to the same
“chemical tail peptide” (C-peptide 1-13) and to a new
heptapeptide (14-20) whose analysis in conjunction with new
results obtained by conventional enzymatic methods [4,5]
necessitated the following correction of the hitherto-accepted
sequence :
NHz
Old
sequence:
- Ser - Thr - Ser - Ser
Revised
sequence:
Numbering:
I
- Asp
- His - Met -
Glu -
NHz
I
- Glu - His - Met - Asp - Ser - Ser - Thr - Ser - 11 - 12 - 13 - 14 - 1 5 - 16 - 17 - I8 -
There is reason to believe that the heptapeptide (14-20)
which has been carved out of ribonuclease-S-peptide with the
aid of cyanogen bromide, is associated with the mode of
action and the active center of the enzyme. K. Hofmann [6]
recently succeeded in synthesizing this heptapeptide. Both
natural as well as synthetic heptapeptide Seem to rearrange
or isomerize under the conditions used for characterization
on the Stein and Moore analyzer, namely citrate buffer
pH < 3.0 and 60°C. This phenomenon is under active
investigation.
[GDCh-Ortsverband Marburg/L. (Germany), July 20th, 19621
[VB 618/53 IE]
The Influence of Chemical Agents on Mental Illness
Cleavage of Proteins without Enzymes
E . Witkop, Bethesda, Md. (U.S.A.)
During the last four years several agents have become known
that permit a more selective cleavage of peptides and proteins
than is possible by enzymatic methods [l]. These novel nonenzymatic cleavage methods are of value in spot-checking
and confirming primary sequences. A special test case in this
connection has been bovine pancreatic ribonuclease. This
enzyme consists of an unbranched chain of 124 amino acids
whose secondary and tertiary structure is determined by four
disulfide bridges. The cautious reductive opening of these
four S-S-bridges in urea solution leads to a complete loss of
secondary and tertiary structure which, however, is reconstituted on slow air-oxidation and removal of urea by dialysis, a process which leads to a fully active enzyme indistinguishable from native ribonuclease [2]. It must be concluded
that the primary sequence of ribonuclease contains directing
groups which help the randomized enzyme in assuming the
proper and thermodynamically favored tertiary structure
necessary for full enzymatic activity. This historic experiment
and similar studies currently conducted in the laboratory of
C. B. Anfinsen attribute to the primary sequence of certain
proteins a novel and far-reaching significance- An interpretation of the directive groups, factors and forces operative
in the randomized chain will only be meaningful, if and when
the position of each individual amino acid in the primary
[I] Cf. B. Witkop, Advances Protein Chem. 16,221 (1961).
[2] F. H. White,jr., J. biol. Chemistry 235, 383 (1960).
604
E. Jucker, Basel (Switzerland)
There are two basic types of psychopharmacological agents :
preparations with psychosedativedepressive action and those
with antidepressive-stimulant action. Some classes of compounds can embrace members with both types of action. The
investigations of the author and his co-workers [7] led to new
thiazepine, thiepine, dibenzocycloheptadiene and dibenzocycloheptatriene derivatives, to derivatives of 1- and 4-azathioxanthene and to the synthesis of the previously unknown
piperidyl-spiro-succinimides;
I
/”\
5,1l-Dihydrobenzo[b]pyrido[2,3.e]1 .4-thizepine derivatives
I
/N\
6,11-DihydrodibenzoIb,eIthiepine derivatives
[3] E. Gross and B. Witkop, J. hiol. Chemistry 237, 1856 (1962).
[4] D. G. Smyth, W. H. Stein, and S. Moore, J. biol. Chemistry
237, 1845 (1962).
[5] J.T. Ports, A . Berger, J. Cooke, and C . E. Anfinsen, J. biol.
Chemistry 237, 1851 (1962).
[6] Unpublished results; personal communication.
[7] A , Ebnbther, F. Gadient, A . Lindenmann, E. Schenker, A.
Stoll, and R . Sues, Pharmaceutical-chemicalResearch Laboratories, SANDOZ A.G., Basel (Switzerland).
Angew. Chem. internat. Edit. J Vol. I (1962) ] No. 11
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