Effect on protein stability of reversing the charge on amino groups.
The amino groups of beta-lactoglobulins A and B, cytochrome c and ribonuclease were progressively converted to acidic groups by reaction with succinic anhydride. The mixtures of modified proteins generated in this way were analyzed by urea-gradient electrophoresis, which separates the molecules on the basis of their net charge and demonstrates visually their urea-induced unfolding transitions. Molecules succinylated to varying extents were resolved by the electrophoresis, so purification of the many modified species was not required. It is demonstrated that accurate estimates of the stability of the folded state of an individual species may be estimated very easily from its urea-gradient electrophoretic pattern. Changes in ionization of the protein upon unfolding may also be detected. The general electrostatic effect of varying the net charge on these proteins was small. Converting the normally basic ribonuclease and cytochrome c to neutral and then to acidic proteins caused the net stabilities of their folded states to vary by no more than a few kJ/mol. However, specific interactions between a few ionized groups appear to be more important in some instances. Succinylation of the 19th, and final, lysine residue of cytochrome c produced unfolding even in the absence of urea, whereas reaction of the first 18 had very little effect. Reaction of the initial amino groups of beta-lactoglobulins A and B produced a small increase in stability in a few instances, a decrease in others; modification of more than about ten groups abruptly caused unfolding in the absence of urea.
D055598:Chemical Phenomena; D002621:Chemistry; D003574:Cytochrome c Group; D004355:Drug Stability; D004586:Electrophoresis; D014644:Genetic Variation; D007782:Lactoglobulins / Q000235:genetics; D008239:Lysine; D011487:Protein Conformation; D012260:Ribonucleases; D013387:Succinic Anhydrides; D013816:Thermodynamics; D014508:Urea
D003574:Cytochrome c Group; D007782:Lactoglobulins; D013387:Succinic Anhydrides; D014508:Urea; D012260:Ribonucleases; D008239:Lysine