True crude assays

The enzyme without the sigma factor, called core polymerase, retains the capability to synthesize RNA, but it is defective in the ability to bind and initiate transcription at true initiation sites on the DNA. In fact when RNA polymerase was first purified from crude extracts it was missing the a factor. The assay for polymerase involved the use of DNA with single-strand nicks. When a DNA template was used that did not have single-strand nicks, this enzyme was not active. This led to a search for a missing factor. When this factor (cr70) was added back to the purified core enzyme and the uncut DNA template, the enzyme was able to bind... 

A full assay involving the preparation of a true boiling point curve and the analysis of fractions and product blends throughout the full range of the crude oil. 

In addition to the whole crude oil tests performed as part of the inspection assay, a comprehensive or full assay requires that the crude be fractionally distilled and the fractions characterized by the relevant tests. Fractionation of the crude oil begins with a true boiling point (TBP) distillation using a fractionating column with an efficiency of 14-18 theoretical plates and operated at a reflux ratio of 5 1 (ASTM D-2892). The TBP distillation may be used for all fractions up to a maximum cut point of about 350°C atmospheric equivalent temperature (AET), but a low residence time in the still (or reduced pressure) is needed to minimize cracking. 

Normally a fraction of the cellular enzyme complement is sufficient to maintain proper function and the regulation of activity is a function of substrate level. In the case of those subjects with partial deficiency of HPRT, it is possible to demonstrate the superiority of the intact cell assay as a measure of the true in vivo situation. In addition, these studies demonstrate the advantage of using an intact cell over the crude lysate in looking for an index of actual enzyme performance in tissues in general. 

It has been suggested many times that these methods have the advantages of simplicity, sensitivity, and ease of execution. Whilst this is possibly true of pure enzyme systems, it is very often far from true of experiments utilising crude tissue extracts. For coupled reactions to give an accurate measure of the primary enzyme reaction, it is essential that the primary product is utilised immediately and completely by the coupled system. Assays involving one coupling enzyme require an excess of coupling enzyme of 100-fold to achieve a 4% accuracy [3]. 

For assays of relatively small amounts of beta-glucuronidase, use 1 mM MUG in Extraction Buffer (see above) with a final reaction volume of 0.5 ml. The reaction is carried out at 37 C. At time=0 and subsequent times, remove samples (e.g., 100 1) and stop the reaction by adding 0.9 ml 0.2 M Na2C03. We have observed that GUS activity remains linear (even in crude extracts) for a very long time, sometimes days. Hence the "time=0" point does not need to be at the moment of addition of extract to substrate. In fact, it is often better to allow the reaction to proceed at 37 C for several minutes to equilibrate and to achieve before the initial time point is taken. Thus, we actually take 5, 15, 25, and 35 minute time points, and always find true linearity is maintained. The resulting slope can therefore be measured independent... 

The lAA-forming activity present in crude buffer extracts could unfortunately not be stabilized to permit isolation of the respective enzyme(s) [2]. It could, however, be attributed to a true oxidase (or a set of oxidases) which was at least group specific for aromatic aldehydes. The assay system did not permit a check for product inhibition by lAA. PAA and the auxin analogue 2,4-D did, however, inhibit indoleacetaldehyde oxidation. 

Assay analyses of whole crudes are done by combining an atmospheric and vacuum distillation run. These two runs when combined will provide a TBP (True Boiling Point Curve). While these batch distillation methods are labor intensive, taking between three to five days, they allow the collection of distillation fractions that can be utilized for testing. While each of the distillations techniques have been standardized by ASTM, cut schemes tend to mimic Refinery product classifications and there is no standardization of the individual inspection formats. Each corporation tends to perform both physical and chemical testing that best meet the needs of their refining operations and product suites. 

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