Previous experiments indicate that enzymes can also exhibit long-lived activity differences at room temperature resulting in a distribution of activities between ostensibly identical molecules, termed “static heterogeneity”. This static heterogeneity arises either from different conformations of the Senegenin enzyme and/or different primary sequences between molecules, with the latter possibility resulting from errors in transcription and translation. When a nascent protein folds, it is possible that each molecule becomes trapped in a local energy minimum and many different local minima, i.e. different conformations, may be populated with each conformation exhibiting a different activity. Moreover, if different enzyme molecules differ in their primary sequence, they could also exhibit a distribution of activities. In this paper, we demonstrate that static heterogeneity of an enzyme population is caused by the existence of many different yet stable conformations. In previous papers, we have described the heterogeneity observed in the activities of Sipeimine individual enzymes, and definitively demonstrated that different initial activities are not caused by surface interactions. There are two possible reasons why the initial activities of the enzymes exhibit such heterogeneity. First, the enzyme molecules may possess different conformations, and consequently different activities, even with the same primary sequence. A second possibility is that the enzymes vary in primary sequence due to errors in transcription and translation. The commercially available b-galactosidase used in this experiment is expressed in E. coli and therefore it is expected that both the transcription and translation processes are not error-free. Transcription errors and translation errors account for 14% and 29% respectively of the protein population having one or more errors. The probability of a b-galactosidase tetramer molecule existing without any errors is only 25%. The rest of the population 
might contain one or more errors in primary sequence. These changes in primary structure might have an influence on the conformational dynamics or energetics, resulting in an altered activity of individual enzymes. In our experimental design, we employed a heating stage to introduce thermal pulses to perturb the conformation of the enzyme molecules. In our system, we observe the activity changes resulting from conformational changes, however we cannot observe individual protein conformations directly. If conformational differences are the cause of static heterogeneity, we would expect that heat pulses would convert some of the molecules into different conformations and we would observe changes in activity from the initial activity state after cooling. On the other hand, if sequence differences are the primary determinant of activity, then heating would not be expected to change the activity. After obtaining the initial activity of the enzyme molecules, five short heating pulses were introduced and the activities were measured after each pulse. When a population of enzymes was exposed to pulses of heat, the activity distributions of the entire population of b-galactosidase molecules narrows slightly after the first two heating pulses. After the second heating pulse, the activity distributions of the populations do not change. Importantly, the average activities of the population after each heating pulse were similar to the average activities of the enzymes prior to heating. When the activities of individual b-galactosidase enzymes were studied, the turnover rates of individual enzymes changed randomly upon introducing a heat pulse and then remained constant during the period between heat pulses.