As a professor of molecular biology and physics, I'm happy to weigh in on this subject. DNA enzymology is a major focus of research in my lab; we study enzymes that recombine (i.e., cut, rearrange, and rejoin) DNA in E. coli and yeast.
i.) Most enzymes progressively lose activity at room and physiological temperature. This is because enzymes are proteins, long-chain molecules that have complex three-dimensional folded shapes. The native 3-d shape is required for catalytic (enzymatic) activity; however, the forces that hold these shapes together are weak and therefore easily overcome by random thermal motions. Increased motion at elevated temperatures not only makes the folded state less stable, but also accelerates the rate of unfolding.
ii.) Unfolding is also made more favorable by dilution. This is because at high concentration proteins have "less room" to unfold due to the larger volume fraction occupied by protein molecules instead of solvent. Once an enzyme solution is diluted, however, the local influence of enzyme molecules on one another is dramatically reduced. The dilution problem is exacerbated by surface inactivation, whereby the greater exposure of enzyme molecules to the surface of a test tube increases the probability that the molecule will stick to a surface and become inactivated.
The way that biochemists generally address the problems associated with dilution is by including high concentrations of an inert protein such as bovine serum albumin (BSA) in their diluted enzyme solutions.
iii.) The duration of enzyme activity one can expect depends on the enzyme and also the solution in which it is suspended. Lifetimes are typically minutes to hours (not necessarily 8 hrs.). Some enzymes, such as ribonuclease A retain activity indefinitely (this is because the protein's 3-d structure is stabilized by covalent sulfur-sulfur bonds). Many, but not all, detergents destabilize folded proteins and if these compounds are present in the enzyme solution they will likely affect the time course of enzymatic activity. However, if the concentration of enzyme is high enough to start with, it may not matter much for periods of an hour or so.
FWIW, long-term storage conditions for our purified enzyme preps involve high concentrations (10 - 100 mg/mL) at -80 deg. C.
Hope this clears up some questions.
i.) Most enzymes progressively lose activity at room and physiological temperature. This is because enzymes are proteins, long-chain molecules that have complex three-dimensional folded shapes. The native 3-d shape is required for catalytic (enzymatic) activity; however, the forces that hold these shapes together are weak and therefore easily overcome by random thermal motions. Increased motion at elevated temperatures not only makes the folded state less stable, but also accelerates the rate of unfolding.
ii.) Unfolding is also made more favorable by dilution. This is because at high concentration proteins have "less room" to unfold due to the larger volume fraction occupied by protein molecules instead of solvent. Once an enzyme solution is diluted, however, the local influence of enzyme molecules on one another is dramatically reduced. The dilution problem is exacerbated by surface inactivation, whereby the greater exposure of enzyme molecules to the surface of a test tube increases the probability that the molecule will stick to a surface and become inactivated.
The way that biochemists generally address the problems associated with dilution is by including high concentrations of an inert protein such as bovine serum albumin (BSA) in their diluted enzyme solutions.
iii.) The duration of enzyme activity one can expect depends on the enzyme and also the solution in which it is suspended. Lifetimes are typically minutes to hours (not necessarily 8 hrs.). Some enzymes, such as ribonuclease A retain activity indefinitely (this is because the protein's 3-d structure is stabilized by covalent sulfur-sulfur bonds). Many, but not all, detergents destabilize folded proteins and if these compounds are present in the enzyme solution they will likely affect the time course of enzymatic activity. However, if the concentration of enzyme is high enough to start with, it may not matter much for periods of an hour or so.
FWIW, long-term storage conditions for our purified enzyme preps involve high concentrations (10 - 100 mg/mL) at -80 deg. C.
Hope this clears up some questions.