A New Method for Protein Hydrolysis with Crystallized . Oxalic Acid

A New Method for Protein Hydrolysis with Crystallized
. Oxalic Acid
Rio de Janeiro
1961

A New Method for Protein Hydrolysis with
Crystallized Oxalic Acid
The well known disadvantages of the conventional methods of protein
hydrolysis prompted us to try "dry heating" of the protein with crystallized
oxalic acid as hydrolyzing agent. This acid, already employed in the pyrohydrolysis
of organic substances by FEIGL[1],was never tried before as a specific
agent for breaking up the peptides bonds. Although LIEKENET AL.[2]observed
a large carbonization and small cleavege of the protein by dry heating with
monochlor acetic acid or with o-chlor benzoic acid, oxalic acid proved to be
an effective and useful hydrolyzing agent.
The protein used in all experiments was a purified commercial grade
of casein; the amino acids standards were obtained from Mann Research
Lab., Inc.
Troll and Cannan's reagents [3] were used for the ninhydrin reaction
and the formaldehyde titration was performed according to the Dunn and
Loshakoff technique [4].
The hydrolysis was followed by paper electrophoresis using a pH 5 O.lM
pyridine-acetic acid buffer.
Casein and the hydrolysing agent were mixed, in the desired proportion,
ground and transferred to test tubes. Some of these tubes were sealed prior
to hydrolysis. After a certain time of heating in oil bath, at 120°C, the tubes
were cooled and the reaction products dissolved in water. It was observed
that the mixture in the open tubes turned liquid after 15 minutes and remained
liquid even after 6 hours of heating, after which it solidified. In the sealed
tubes it remained liquid even after 24 hours of heating. After the reaction
the sealed tubes had to be cooled in a dry ice-acetone mixture before being
opened. This was necessary to avoid the violent explosions of these tubes.
For the subsequent tests it became necessary to eliminate the acid,
and the best result was obtained by utilyzing powdered CaC0 3 • This has
the advantage of not increasing the volume of the liquid to any considerable
extent, and when used in excess, remains insoluble. Other methods of eliminating
the oxalic acid, such as ion-exchange resins were also tried giving, however,

poor results. The calcium oxalate (which contrary to BaSOo! adsorbs very
little of the amino acids) was filtered off and washed a few times with hot water.
The filtrates were combined and made up to a known volume; from this solution
aliquots were taken for formaldehyde titration, ninhydrin reaction and paper
electrophoresis.
In order to have a better understanding of the hydrolitic process,
other hydrolitic agents such as anhydrous oxalic acid and crystallized salts
were used as shown in Table 1.
Protein Time of
HYDROLYSING AGENT hydrolysing hydrolysis Ninhydrin value
agent ratio in hours
Oxalic acid (2H20) ................ 1:1 6 0,735 (a)
Same ............................. 1:2 6 1,570 (a)
Same ............................ 1:5 6 2,180 (a)
Same ............................. 1:10 6 2,600 (a)
Same ............................ 1:20 6 2,660 (a)
Same ............................ 1:50 6 2,800 (a)
Oxalic acid (anhydrous) ........... 1:10 6 0,000 (a)
Sodium sulphate (10H 2 0) .......... 1:10 24 0,040 (b)
Sodium acetate (3H20) ............ 1:10 24 0,095 (b)
The amino acids recovery (Table II), was measured by the ninhydrin
reaction after 6 hours of hydrolysis with oxalic acid. In the particular case of
tryptophane it was also determined spectrophotometric ally at 280 m J.L.
AMINO ACIDS I Recovery
(%)
Arl;inine .... .. .... . ... . .... 60
Serine ... ... . . . ....... ... . 65
Cysteine ...... ... . . . . ... . . . . 76
Threonine ...... .. . . ... .... . . . 78
Tryptophan ........ .... . . ... 90
Phenylalanine ....... .. . . . . . 100
Tyrosine .... .. . .. . .... . ... 100

process,
:ed salts
As can be seen in the Tables I and II and Figures 1 and 2, the dry
heating of a protein with crystallized oxalic acid causes the breaking of the
peptides linkages with liberation of amino acids. This result was confirmed
by paper electrophoresis and paper chromatography.
The hydrolysis in sealed tubes is more complete and less destructive
than m open tubes.
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Fig. 1 -- Casein hydrolysis with crystallized oxalic acid. The hydrolysis was followed with
the Sorensen titration (4).
Experiments showed (TABLE I) that the hydrolysing agent must
be an acid and that the presence of water is necessary, as the anhydrous acid
and the neutral salts had no hydrolytic effect. The best proportion of reagents
for hydrolysis, were found to be 1 part of protein to 10 parts of oxalic acid,
because it combined rapid hydrolysis with greater facility in the elimination
of the acid. As can be seen in the TABLE II the present process, partially
destroys some of the acids. However tryptophane, which is completely
destroyed in the course of the conventional methods of acids hydrolysis, shows
a 90% recovery after hydrolysis with oxalic acid. The speed of hydrolysis,
the easiness of the elimination of the hydrolyzing agent and the preservation
of some of the easily destroyed amino acids are the advantages of the present
method of hydrolysis.

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Fig. 2 - Casein hydrolysis with 6N HCI and crystallized oxalic acid. The hydrolysis were
followed with the ninhydrin reaction (3).
A new process for the hydrolysis of proteins was developed, based on the dry heating,
at 120°C, of the proteins, with cristallized oxalic acid. The influence of various factors, such
as the duration of hydrolysis, temperature and protein-oxalic acid proportions were studied;
parallel experiments in open and sealed test tubes were made, as well as conventional hydrolysis
with acid solutions. All tests were followed up with Sorensen and ninhydrin reactions and
paper electrophoresis.
The stability of several amino acids in regard to this type of hydrolysis was investigated,
a.~d the conclusions reached were in favour of the present method as it does not destroy the amino
acids ordinarily decomposed in the course of acid hydrolysis and, also, allows an easy elimination
of the hydrolysing agent (oxalic acid.)
[1] FEIGL, F., (1958), Ang. Chernie, 70, 166.
[2] DUNN, M. S. AND LOSHAKOFF, A., (1936), ]. BioI. Chern., 113, 359.
[3] TROLL, W. AND CANNAN, K. K., (1953), ]. BioI. Chern., 200, 803.
[4] LIEKEN, FAND AND TANDLER, R., (1959). Sci. Pharrn., 27, 82.