A New Method for Protein Hydrolysis with Crystallized . Oxalic Acid

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,