Analysis of free sulfhydryl groups and disulfide bonds in Na⁺,K⁺‐ATPase

Abstract

The content of free SH groups and disulfide bonds in the purified pig kidney Na+,K+-ATPase was determined by ammetric titration with silver nitrate. In the native enzyme, most of the free SH groups are masked due to their location in the polypeptide chain regions poorly accessible to SH reagents. Denaturation with 5% SDS and 8 M urea makes these regions accessible thus revealing 22 free SH groups/mol of the protein. After complete blocking of free SH groups with silver ions, 8 SH groups/mol of the protein are being released upon sulfitolysis which indicates the presence of four disulfide bonds in the enzyme. At least one disulfide bridge is located in the a-subunit whereas the @-subunit contains three disulfide bonds.

Na+,K+-ATPase as the Target Enzyme for Organic and Inorganic Compounds

“…A decrease in Na + ,K + -ATPase activity was reported in vesicles of rabbit brain choroids plexus [129]. The increase in free –SH group content may imply that phenoxyl herbicides damage the enzyme molecule structure by disruption of disulfide bridges [130]. These disruptions lead to enzyme inactivation.…”

Kinetic and thermodynamic analysis of ultra-high pressure and heat-induced denaturation of bovine serum albumin by surface plasmon resonance

“…Similar to most other antigen-antibody interactions, the binding of an antigen is enthalpically driven throughout the physiological temperature range. Structural flexibility of the antigen-antibody site is key to the antigen binding activity [20]. Burgen et al [21] suggested that such a conformational flexibility would result in a faster rate of association and dissociation, a mechanism of recognition tagged the "induced fit" model.…”

“…A significant amount of free SH groups in the native globular conformation were masked to attack, due to their location in poorly accessible regions of the folded structure. Therefore, the level of SH groups could be considered as an indication of protein unfolding or denaturation [20].…”

Quick measurement of protein sulfhydryls with Ellman's reagent and with 4,4′-dithiodipyridine

“…As a consequence, the quantitative measurement of SH groups is a routine task in many applied disciplines where a quick and easy method is much preferred. Electrochemical [3,4] and fluorimetric assays [5,6,7,8] of protein sulfhydryls are very sensitive and accurate but they involve lengthy procedures (complete proteolysis, electrolysis, HPLC separation). Sensitivity and reliability are also good in an enzymatic method where protein sulfhydryls are first reacted with cystamine and the released cysteamine cleaves papain-S-S-CH 3 , yielding active papain-SH which is then assayed with a fluorigenic substrate [9].…”