Deforming agent

Apply to the column 1.0ml of peptide solution (dissolved in equilibration buffer) to be reduced. Normally, small peptides (molecular weight less than or equal to that of insulin) require no deforming agent (denaturant) such as guanidine to be completely reduced. 

Apply to the column 1.0ml of protein solution (dissolved in equilibration buffer-2) to be reduced. The inclusion of a denaturant in the solution deforms the protein structure so that inner disulfides are available to the immobilized reductant. Without the presence of guanidine or another deforming agent (i.e., urea, SDS, etc.), only partial reduction of the protein is possible. 

For small molecules containing sulfhydryls or for low-molecular-weight peptides containing cysteine residues, modification may proceed without deforming agents. However, for intact proteins containing both disulfides and free sulfhydryls, a denaturant and a disulfide reducing... 

Tyrosine contains a phenolic side chain with a pKa of about 9.7—10.1. Due to its aromatic character, tyrosine is second only to tryptophan in contributing to a protein s overall absorptivity at 275—280 nm. Although the amino acid is only sparingly soluble in water, the ionizable nature of the phenolic group makes it often appear in hydrophilic regions of a protein—usually at or near the surface. Thus tyrosine derivatization proceeds without much need for deforming agents to further open protein structure. 

Although TCEP is capable of rapidly and quantitatively reducing simple organic disulfides in solution, it requires the presence of a deforming agent to reduce fully all disulfides in proteins. Without opening up the internal disulfides in many protein molecules, TCEP will not be able to reduce them. For complete reduction of IgG, it was found that 20 mM TCEP and 5 min of boiling was needed (Hines, 1992). Partial reduction, however, is possible of some more accessible disulfides in aqueous buffers at room temperature. 

The resultant culture was inoculated to a medium containing soluble starch (5%), peanut powder (0.5%), dried yeast (0.5%), gluten meal (0.5%), calcium carbonate (0.1%) and Adekanol (deforming agent. Trade Mark, maker Asasi Denka Co.) (0.1%) (150 liters) in a 200-liter jar-fermentor, which had been sterilized at 120°C for 20 min in advance, and cultured at 30C for 4 days under aeration of 150 liters/minutes and agitation of 250 rpm. 

Enormous quantities of these two compounds were used in an approximately 1 1 mixture as the defoliant Agent Orange during the Vietnam War. Some samples of 2,4,5-T were shown to be teratogenic (a fetus-deforming agent), and its use has been banned in the United States. 

---