Protein cold inactivation

The prompt removal of acetyl glutamate from the preincubation mixtures, by dialysis at room temperature or by precipitation of the protein with ammonium sulfate, prevented the cold inactivation. The presence of ammonium sulfate, in concentrations as high as 0.17M, did not protect the enzyme against cold inactivation (at higher molarities of the salt, precipitation began to occur). Once the cold inactivation had taken place, the enzymatic activity could not be restored either by precipitation with ammonium sulfate, or by reincubation with acetyl glutamate or with ATP and Mg+2. 

Other Physical Measurements. Viscosity measurements did not show differences between samples treated with acetyl glutamate and controls, before and after storage in the cold (35). Extensive series of ultracentrifuge measurements did not clarify the phenomenon the latter studies were conducted at several protein and acetyl glutamate concentrations, and at temperatures varying from 5° to 20° only after cold inactivation for 40 hours were marked changes noticeable. 

HMG-CoA reductase, the enzyme that catalyses the formation of mevalonate [MVA, (2)] from HMG by an irreversible reaction that is considered rate-limiting with respect to the formation of cholesterol, has received much attention. Details of the purification of the enzyme from chicken liver and baker s yeast are available,15 and the solubilized enzyme from rat liver microsomes is readily and reversibly inactivated at temperatures below 19°C.16 Cold-inactivation is an uncommon phenomenon, and all the enzymes that have been found to exhibit this behaviour have been soluble proteins. Native HMG-CoA reductase is a particulate enzyme that is probably bound to protein or lipid of the microsomal membrane, although it is not known whether the solubilized enzyme contains a lipid component. Microsomal reductase is not cold-sensitive, and the cold-inactivation of the solubilized enzyme can be completely prevented by addition to the preparation of NADP+ or (more effectively) of NAD PH.17... 

In its cold-inactivated state, a protein is not markedly susceptible to pH changes or salt effects and is not subject to aggregation. 

Leghemoglobin function Cold inactivation of Fe protein Activation energy change at 20° Nomenclature... 

To study the effect of pH and temperature, 0.25 ml of the lignin peroxidase diluted with water was mixed with 0.75 ml of buffer, pH 3.0 - 7.0 and incubated at temperatures of30- 70°C. The protein concentration of the incubation mixture was 50 ig/ml. After various incubation times (0 - 27 h) the inactivation was stopped by adding 9 ml of cold 0.33 M tartrate buffer, pH 3.0. 

The major manufacturing process for plasma-derived products is the fractionation of human plasma, the liquid part of blood, to remove the minute amounts of plasma proteins present in each unit of plasma. To make the process commercially successful, very large quantities of plasma are mixed together and then fractionated. Dr. Elias Cohn developed the initial process in the early 1940s at Harvard University using differential cold alcohol precipitation. Essentially the same process (as modified by Oncley) is used today, with the addition of more rigorous viral inactivation techniques to increase safety. The conditions have been set to both efficiently fractionate the protein and to maximize viral partition, inactivation, or removal. Considering the... 

We typically do not use protease inhibitors. The combination of the lysis buffer with its reducing ability, the chaotropic effects of the urea and the surfactant, and the cold temperature seems to inactivate proteolytic activity. We also do not perform any steps requiring room temperature or protein activity (such as the DNAse-RNAse treatment found in some protocols). Furthermore, the presence of the inhibitors may sometimes interfere with the fluorescent labeling. The sample cups on the lEF gel have about 100-pl maximum capacity. However, if necessary, more volume can be handled by ordering more sample cup holder bars separately from the dry strip kit and used to spread one sample between several cups. Because IFF is a focusing technique, the sample does not necessarily have to be applied in exactly the same spot. The dye synthesis is detailed elsewhere. The dyes are not commercially available as of the time of this writing. 

Cold denaturation (unfolding, dissociation, inactivation) is 100% reversible, even at high protein concentrations. 

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