The Reversible Heat Denaturation of Chymotrypsinogen

Abstract

Although the process of the reversible denaturation of proteins is one of the most challenging problems of protein chemistry, this field has been relatively dormant for about 10 years. 1 Recently, the excellent study by Kunitz (1) of the reversible heat denaturation of crystalline soy bean trypsin inhibitor has given rise to a revival of interest in this problem. The present investigation has been made as a part of the general study of the pancreatic proteases which has been carried out in this laboratory.The reversible heat denaturation of trypsin has been studied by Northrop (2), by Anson and Mirsky (3), and by Kunitz and Northrop (4) and the reversible heat denaturation of chymotrypsin has been studied by Kunitz and Northrop (5). Investigations of reversible denaturation with proteolytic enzymes may be complicated by the fact that over a large portion of the pH scale the native protein is enzymatically active and digests the denatured protein. For this reason, chymotrypsinogen, which has also been found to undergo reversible heat denaturation but which has no known enzymatic activity, was selected for the present study. MethodsCrystalline chymotrypsinogen was prepared and recrystaUized eight times by the method of Kunitz and Northrop (6). The purity of this preparation was tested by measuring its solubility at 25°C. in 0.2 ~ acetate buffer of pH 5.08 containing 0.5 saturated ammonium sulfate. The technique of Kunitz (7) was used for these measurements. The solubility diagram was that of a single component with a solubility of 0.350 rag. of protein per ml. even in the presence of an eight/old excess of solid phase.The crystalline protein was dissolved in 3 volumes of water at pH 3.0 and was dialyzed with stirring against frequent changes of 0.001 N HC1 until negative tests for both ammonia and sulfate were found in a 12 hour dialysate.

CHANGES IN HYDRATION, SOLUBILITY AND CHARGES OF MUSCLE PROTEINS DURING HEATING OF MEATa

“…Stauff (61), however, suggests the possibility that the first step is an intramolecular swelling and a loosening of protein structure rather than a real unfolding (16). usually the first step of denaturation is considered to be reversible, the second, irreversible (19).…”

Early days of protein hydrogen exchange: 1954–1972

“…Slow denaturation (hours) of relatively complex proteins is characteristic of these earlier studies, which were interpreted in terms of a two‐state equilibrium between native and denatured forms. A 1951 study of the thermal denaturation of chymotrypsinogen A by Eisenberg and Schwert15 emphasizes its reversibility and rapidity (∼seconds) but misses the later point that denaturation is produced by unfolding. These authors found a huge activation enthalpy for denaturation and interpreted15 it as the result of breaking many interactions between the water lattice and the native protein.…”

The Effect of Cookery on Muscle Proteins and Meat Palatability: A Review

“…The overall process results in a coagulation effect of the proteins, which is the second step in the hypothesis of protein denaturation by Wu (1931). Eisenberg and Schwert (1954) reported that the first step of denaturation is reversible, and thus, the protein can be returned to its native conformation, while the second step is not reversible. Stauffs (1956) reasoning for this was that the first step may be more of an intramolecular swelling and a loosening of the protein structure rather than a n actual unfolding.…”