In extending our investigation of the enzymatic synthesis of protein

to the synthesizing action of commercial trypsin (1) the findings

of Henriques and Gjaldbäk (2) were reviewed. In their experiments

on plastein formation by trypsin, these authors observed

a curious simultaneous hydrolysis and synthesis. This observation

is confirmed

Borsook, Henry

Wasteneys, Hardolph

Caltech Authors - Main

THE ENZYMATIC SYNTHESIS OF PROTEIN. V. 
A NOTE ON THE SYNTHESIZING ACTION OF TRYPSIN. 
BY HARDOLPH WASTENEYS AND HENRY BORSOOK. 
(From the Department of Biochemistry, University of Toronto, Toronto, 
Canada.) 
(Received for publication, February 14, 1925.) 
In extending our investigation of the enzymatic synthesis of pro- 
tein to the synthesizing action of commercial trypsin (1) the find- 
ings of Henriques and Gjaldbak (2) were reviewed. In their experi- 
ments on plastein formation by trypsin, these authors observed 
a curious simultaneous hydrolysis and synthesis. This observa- 
tion is confirmed. 
It was first necessary t,o determine the optimum pH for tryptic 
synthesis. 
Solutions were made up as recorded in Table I, using a peptic 
hydrolysate of albumin as substrate, and adding the amounts of 
acid, alkali, or water necessary to obtain the required pH and a 
constant concentration of digest. 
The trypsin employed was prepared from “Difco” trypsin. A 
10 per cent solution of the commercial product was adjusted to 
pH 6.5, centrifuged, filtered, and then precipitated with 9 volumes 
of alcohol. The precipitate was dried, first with alcohol and ether, 
and finally in a vacuum desiccator. A fine, whit.e product was 
obtained. The solution was adjusted to pH 6.5, because at that 
hydrogen ion concentration, as Northrop showed (3), the auto- 
destruction of trypsin is least rapid., Out of each of the above 
mixtures two 10 cc. portions were pipetted into 50 cc. Erlen- 
meyer flasks containing 0.10 gm. of the prepared trypsin, dissolved 
in 1 cc. of water. The solutions were thoroughly stirred and set 
away with chloroform in the incubator at 37°C. 
Of the sixteen flasks, eight were analyzed for protein at the end 
of 3 days, and the other eight at the end of 5 days. 
575 
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576 Synthesizing Action of Trypsin 
At the end of 1 hour, the contents of Flasks 1,2,3, and 4 showed 
precipitates and were almost stiff. Flasks 6 and 7 were immov- 
able transparent jellies. Flask 5 contained some precipitate, but 
was also somewhat gelatinous. 
At the end of 2 days, Flask 7 (pH 9.0) contained a large number 
of white particles dispersed through the jelly. These, under the 
microscope, were seen to be composed of crystals resembling those 
TABLE I. 
No. 
20 
20 
20 
20 
20 
20 
20 
20 
1 2.0 NHCI. 2.0 N NaOH Hz0 PH 
cc. 
1.5 
0.9 
~ cc. 
0.6 
2.0 
3.3 
5.0 
6.4 
TABLE II. 
Effect of CH on Tryptic Synthesis. 
5.4 
5.8 
6.5 
7.1 
7.7 
8.3 
9.0 
9.9 
cc. 
5.5 
6.1 
7.0 
6.4 
5.0 
3.7 
2.0 
0.6 
PH 
5.4 
5.8 
6.5 
7.1 
7.7 
8.3 
9.0 
9.9 
Protein N in per cent of total N. 
3 days. 
12.8 
13.1 
11.5 
9.1 
7.6 
8.1 
6.4 
5.1 
5 days. 
14.0 
15.4 
13.2 
7.9 
6.6 
6.2 
6.7 
of tyrosine and cystine, and further evidence that they consisted 
of these amino acids was obtained from their solubilities, Millon’s 
and sulfur tests. They failed to give the biuret reaction. The 
particles were present in all the alkaline mixtures, diminishing in 
number with decrease of alkalinity. The acid solutions contained 
the typical protein precipitates found in peptic synthesis, and no 
precipitated amino acids. 
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H. Wasteneys and H. Borsook 577 
At the end of the period of incubation the mixtures were diluted 
to 100 cc. and 40 cc. of each of the resulting suspensions were 
pipetted into 10 cc. of 2 per cent trichloroacetic acid. In this con- 
centration of acid, tyrosine and cystine are quite soluble. The 
mixtures of diluted trichloroacetic acid were allowed to stand for 
24 hours in order to allow the precipitated amino acids to redis- 
Per cent 
I- 
IO 
FIG. 1. Relation between CH and the synthesizing action of trypsin. 
solve, and the resolution was facilitated by frequent rubbing and 
shaking. The precipitated protein was then filtered off and the 
total nitrogen of the filtrate determined by the macro Kjeldahl 
method. 
The difference between this and the total nitrogen of the original, 
diluted digest was a measure of the protein synthesized. The 
results of the analyses of the 3 and 4 day series are stated in Table 
II and Fig. 1. 
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578 Synthesizing Action of Trypsin 
The substance synthesized has been called protein because it 
possesses the general properties of protein. It resembles the pro- 
tein synthesized by pepsin from a similar peptic hydrolysate of 
egg albumin. It is very soluble in dilute alkali, less so, but 
still fairly soluble, in dilute acid. It gives the biuret reaction and 
is precipitated from its solution in acid by trichloroacetic acid. 
The optimum pH for tryptic synthesis, as Fig. 1 shows, is in the 
neighborhood of 5.7. From the location of the optimum the 
synthesizing enzyme is clearly not pepsin. In all the alkaline 
solutions, as Henriques and Gjaldbak found, a simultaneous 
hydrolysis ensues. Whether or not both these reactions are pro- 
moted by the same enzyme cannot be decided by these experiments. 
The hydrolysis is strikingly brought out by the greater steepness 
of the 5 day curve than that of the 3 day curve. Whereas all the 
flasks on the acid side of pH 7.0 contained larger amounts of pro- 
tein at the end of 5 days than at the end of 3, on the alkaline side 
they were found to contain consistently less. The synthesis is a 
more rapid reaction than the hydrolysis, which is to be expected 
in solutions of such high concentrations. The hydrolysis follow- 
ing later, and continuing for a longer time, to some extent reverses 
the synthesis that has occurred. 
SUMMARY. 
1. The optimum hydrogen ion concentration for tryptic synthe- 
sis of protein in a peptic digest of egg albumin is shown to be in 
the neighborhood of pH 5.7. 
2. As had been observed previously by Henriques and Gjald- 
b&k, in neutral and alkaline reactions, an hydrolysis also occurs 
simultaneously with the synthesis. 
BIBLIOGRAPHY. 
1. Wasteneys, H., and Borsook, H., J. Biol. Chem., 1924-25, Ixii, 15,633,675. 
2. Henriques, V., and GjaldbLk, J. K,, 2. physiol. Chem., 1912, lxxxi, 439. 
3. Northrop, J. H., J. Gen. Physiol., 1921-22, iv, 261. 
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English

The Enzymatic Synthesis Of Protein. V. A Note On The Synthesizing Action Of Trypsin

THE ENZYMATIC SYNTHESIS OF PROTEIN. V. 
A NOTE ON THE SYNTHESIZING ACTION OF TRYPSIN. 
BY HARDOLPH WASTENEYS AND HENRY BORSOOK. 
(From the Department of Biochemistry, University of Toronto, Toronto, 
Canada.) 
(Received for publication, February 14, 1925.) 
In extending our investigation of the enzymatic synthesis of pro- 
tein to the synthesizing action of commercial trypsin (1) the find- 
ings of Henriques and Gjaldbak (2) were reviewed. In their experi- 
ments on plastein formation by trypsin, these authors observed 
a curious simultaneous hydrolysis and synthesis. This observa- 
tion is confirmed. 
It was first necessary t,o determine the optimum pH for tryptic 
synthesis. 
Solutions were made up as recorded in Table I, using a peptic 
hydrolysate of albumin as substrate, and adding the amounts of 
acid, alkali, or water necessary to obtain the required pH and a 
constant concentration of digest. 
The trypsin employed was prepared from “Difco” trypsin. A 
10 per cent solution of the commercial product was adjusted to 
pH 6.5, centrifuged, filtered, and then precipitated with 9 volumes 
of alcohol. The precipitate was dried, first with alcohol and ether, 
and finally in a vacuum desiccator. A fine, whit.e product was 
obtained. The solution was adjusted to pH 6.5, because at that 
hydrogen ion concentration, as Northrop showed (3), the auto- 
destruction of trypsin is least rapid., Out of each of the above 
mixtures two 10 cc. portions were pipetted into 50 cc. Erlen- 
meyer flasks containing 0.10 gm. of the prepared trypsin, dissolved 
in 1 cc. of water. The solutions were thoroughly stirred and set 
away with chloroform in the incubator at 37°C. 
Of the sixteen flasks, eight were analyzed for protein at the end 
of 3 days, and the other eight at the end of 5 days. 
575 
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576 Synthesizing Action of Trypsin 
At the end of 1 hour, the contents of Flasks 1,2,3, and 4 showed 
precipitates and were almost stiff. Flasks 6 and 7 were immov- 
able transparent jellies. Flask 5 contained some precipitate, but 
was also somewhat gelatinous. 
At the end of 2 days, Flask 7 (pH 9.0) contained a large number 
of white particles dispersed through the jelly. These, under the 
microscope, were seen to be composed of crystals resembling those 
TABLE I. 
No. 
20 
20 
20 
20 
20 
20 
20 
20 
1 2.0 NHCI. 2.0 N NaOH Hz0 PH 
cc. 
1.5 
0.9 
~ cc. 
0.6 
2.0 
3.3 
5.0 
6.4 
TABLE II. 
Effect of CH on Tryptic Synthesis. 
5.4 
5.8 
6.5 
7.1 
7.7 
8.3 
9.0 
9.9 
cc. 
5.5 
6.1 
7.0 
6.4 
5.0 
3.7 
2.0 
0.6 
PH 
5.4 
5.8 
6.5 
7.1 
7.7 
8.3 
9.0 
9.9 
Protein N in per cent of total N. 
3 days. 
12.8 
13.1 
11.5 
9.1 
7.6 
8.1 
6.4 
5.1 
5 days. 
14.0 
15.4 
13.2 
7.9 
6.6 
6.2 
6.7 
of tyrosine and cystine, and further evidence that they consisted 
of these amino acids was obtained from their solubilities, Millon’s 
and sulfur tests. They failed to give the biuret reaction. The 
particles were present in all the alkaline mixtures, diminishing in 
number with decrease of alkalinity. The acid solutions contained 
the typical protein precipitates found in peptic synthesis, and no 
precipitated amino acids. 
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H. Wasteneys and H. Borsook 577 
At the end of the period of incubation the mixtures were diluted 
to 100 cc. and 40 cc. of each of the resulting suspensions were 
pipetted into 10 cc. of 2 per cent trichloroacetic acid. In this con- 
centration of acid, tyrosine and cystine are quite soluble. The 
mixtures of diluted trichloroacetic acid were allowed to stand for 
24 hours in order to allow the precipitated amino acids to redis- 
Per cent 
I- 
IO 
FIG. 1. Relation between CH and the synthesizing action of trypsin. 
solve, and the resolution was facilitated by frequent rubbing and 
shaking. The precipitated protein was then filtered off and the 
total nitrogen of the filtrate determined by the macro Kjeldahl 
method. 
The difference between this and the total nitrogen of the original, 
diluted digest was a measure of the protein synthesized. The 
results of the analyses of the 3 and 4 day series are stated in Table 
II and Fig. 1. 
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578 Synthesizing Action of Trypsin 
The substance synthesized has been called protein because it 
possesses the general properties of protein. It resembles the pro- 
tein synthesized by pepsin from a similar peptic hydrolysate of 
egg albumin. It is very soluble in dilute alkali, less so, but 
still fairly soluble, in dilute acid. It gives the biuret reaction and 
is precipitated from its solution in acid by trichloroacetic acid. 
The optimum pH for tryptic synthesis, as Fig. 1 shows, is in the 
neighborhood of 5.7. From the location of the optimum the 
synthesizing enzyme is clearly not pepsin. In all the alkaline 
solutions, as Henriques and Gjaldbak found, a simultaneous 
hydrolysis ensues. Whether or not both these reactions are pro- 
moted by the same enzyme cannot be decided by these experiments. 
The hydrolysis is strikingly brought out by the greater steepness 
of the 5 day curve than that of the 3 day curve. Whereas all the 
flasks on the acid side of pH 7.0 contained larger amounts of pro- 
tein at the end of 5 days than at the end of 3, on the alkaline side 
they were found to contain consistently less. The synthesis is a 
more rapid reaction than the hydrolysis, which is to be expected 
in solutions of such high concentrations. The hydrolysis follow- 
ing later, and continuing for a longer time, to some extent reverses 
the synthesis that has occurred. 
SUMMARY. 
1. The optimum hydrogen ion concentration for tryptic synthe- 
sis of protein in a peptic digest of egg albumin is shown to be in 
the neighborhood of pH 5.7. 
2. As had been observed previously by Henriques and Gjald- 
b&k, in neutral and alkaline reactions, an hydrolysis also occurs 
simultaneously with the synthesis. 
BIBLIOGRAPHY. 
1. Wasteneys, H., and Borsook, H., J. Biol. Chem., 1924-25, Ixii, 15,633,675. 
2. Henriques, V., and GjaldbLk, J. K,, 2. physiol. Chem., 1912, lxxxi, 439. 
3. Northrop, J. H., J. Gen. Physiol., 1921-22, iv, 261. 
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The Enzymatic Synthesis Of Protein. V. A Note On The Synthesizing Action Of Trypsin

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