Labile proteins

These high performance size exclusion separations of alpha-chymotrypsin SI and myosin light chains compare favorably with those achieved by ion exchange chromatography but require only a fraction of the time to accomplish. Furthermore, the very short retention times allow for separation of these labile proteins at room temperature, whereas operation at 0-40 C would otherwise be mandatory to avoid the loss of enzymatic activity. 

M.L. Ferrer, F. del Monte, and D. Levy, A novel and simple alcohol-free sol-gel route for encapsulation of labile proteins. Chem. Mater 14, 3619-3621 (2002). 

Special attention has been paid by sevo groups to cloning xylanase genes from thermophilic microorganisms as a source of thermostable xylanases. Cloning of such genes in mesophilic recepients offers a convenient way to purify xylanases simply by a heat denaturation of the more heat-labile proteins of the host (54). Applications are limited to those enzymes that possess thermostability considerably higher than the majority of the host cell proteins. 

In contrast to the heat-labile proteins, DNA is relatively stable and can be tested in samples heated up to 120 °C (Lenstra, 2003). It is, unlike proteins, less affected by physiological conditions, environmental factors, storage, and processing (Shaw et ah, 2002). DNA sequences are now widely used for species identification in DNA barcoding (Ratnasingham and Hebert,... 

This system allows the separation of alkali-labile proteins (e.g., acylphosphate phosphoproteins) under denaturating conditions according to their molar mass. Despite the low acrylamide concentration (%T = 5.61, %C = 3.61), the separation force is remarkable. Because it is a SDS-containing system, the migration is from to + despite the low pH. 

The activator for the hydroly sis of G 12 catalyzed by /3-hexosaminidase A was described by Hechtman277 and Hechtman and LeBlanc.278 The GM2-specific activator was purified over 100-fold from human liver, and was identified as a heat-labile protein. This activator did not stimulate the hydrolysis of asialo-G 12 catalyzed either by hexosaminidase A or B. The molecular weight of this activator was determined to... 

Other agents besides ammonium sulfate or other salts can be employed to effect precipitation. As an example, for add-stable enzymes such as are often encountered in proteins from Lactobacilli, responsible for spoiling milk, low pH conditions often lead to precipitation of unwanted proteins while the target protein survives (for an example, see Riebel, 2002). Likewise, heat (typically 1 h at 50-60°C) can be employed to separate heat-labile proteins from more stable ones. 

Castor Ricinus communis (Euphorbiaceae) seed 35-55 ricinoleic (80-90), oleic (4-9), linoleic (2-7), palmitic (2-3), stearic (2-3) emollient base, purgative, soap manufacture Castor seeds contain the highly toxic, but heat-labile protein ricin (see page 434)... 

Keil, R. G., and D. L. Kirchman. 1994. Abiotic transformation of labile protein to refractory protein in sea water. Marine Chemistry 45 187-196. 

Interestingly, the human genome project has indicated the presence of a hornerin-like gene close to profilaggrin on chromosome lq21, but its expression has not been reported or studied in man to date. It remains to be established whether, in otherwise healthy skin, an age-related decline in the ability to synthesize profilaggrin can be compensated for by synthesis of another proteolytically labile protein. 

In 1979, Simpson [24] postulated that the outer mitochondrial membrane was the site of action of a labile protein factor, necessary to facilitate the transport of cholesterol, and Privalle et al. [19] provided evidence to support the notion that transference of cholesterol from the outer to the inner membrane required an agent that is cycloheximide dependent. When rats were ether-stressed in vivo and cholesterol SCC was deliberately inhibited, cholesterol accumulated in the adrenal mitochondria, most (90%) of this being associated with the inner membrane cytochrome P-450scc. After administration of aminoglutethimide to rats to block SCC, there was a two-fold increase in inner membrane cholesterol, while cycloheximide abolished this increase. Thus, it appears that cholesterol accumulates in the inner mitochondrial membrane as a result of stress and that transference from outer to inner membrane requires a protein factor. 

Different industries pose different challenges to the protein formulator. Many feed enzymes are sold as formulated liquid concentrates. In this case, the major requirements for a liquid formulation are enzymatic stability and preservation against microbial growth. It is sometimes not appreciated that the dominant factor affecting enzyme stability is the intrinsic stability of the enzyme itself formulation can do very little to correct for a structurally labile protein. Therefore, it is advisable to make stability an important criterion of the initial enzyme screening process. 

Factor V deficiency is cansed by prodnction of a labile protein. 

Macfarlane, D.E. (1983) Use of benzyldimethyl-n-hexadecylammonium chloride ( 16-BAC ), a cationic detergent, in an acidic polyacrylamide gel electrophoresis system to detect base labile protein methylation in intact cells. Anal. Biochem. J32, 231-235. 

Cost and convenience are the final considerations to be made. During purification the activity of the desired enzyme should be monitored after each step. The ease with which this can be done largely dictates how closely the activity is in fact monitored and how much the purification procedure is lengthened owing to resulting losses of time. These time losses may be significant because as the time required to purify a labile protein increases so does the probability of its inactivation. 

---