Spectrin assay

Figure 3. Critical concentration behavior of actin self-assembly. For the top diagram depicting the macroscopic critical concentration curve, one determines the total amount of polymerized actin by methods that measure the sum of addition and release processes occurring at both ends. Examples of such methods are sedimentation, light scattering, fluorescence assays with pyrene-labeled actin, and viscosity measurements. Forthe bottom curves, the polymerization behavior is typically determined by fluorescence assays conducted under conditions where one of the ends is blocked by the presence of molecules such as gelsolin (a barbed-end capping protein) or spectrin-band 4.1 -actin (a complex prepared from erythrocyte membranes, such that only barbed-end growth occurs). Note further that the barbed end (or (+)-end) has a lower critical concentration than the pointed end (or (-)-end). This differential stabilization requires the occurrence of ATP hydrolysis to supply the free energy that drives subunit addition to the (+)-end at the expense of the subunit loss from the (-)-end.

Tvo additional peripheral membrane proteins anchor the spectrin filaments to the cytoplasmic side of the erythrocyte membrane. One of these polypeptides, the 210-kDa ankyrin protein, binds both a single spectrin molecule and the chloride-bicarbonate anion-exchange protein discussed previously. The second of these polypeptides, actin, is capable of binding several molecules of spectrin. Since actin is able to associate with more than a single spectrin monomer, it acts as a branch point for the spectrin protein as the membrane skeleton or matrix is assembled (see Fig. 13-1). In this experiment, you will determine the concentration of total protein in the erythrocyte membrane through the use of the Folin-Ciocalteau assay. 

The TBA method has been used not only to assay glycosylated hemoglobin, but also almost exclusively for the following glycosylated proteins plasma albumin (D22, El, NIO, M12), plasma proteins and albumin (K6), capillary whole-blood protein collected on filter paper (L14), erythrocyte spectrin (M13), skin collagen (P2), lens basement membrane (M5), and crystallins (M28). In assays on hair and epidermal keratin, cyclohexanone extraction of the color developed with TBA and reading the absorbance of the cyclohexanone layer at 433 nm has provided increased sensitivity and accuracy (T4). A possible objection to the procedure is the use of oxalic and thiobarbituric acids, which are potentially toxic chemicals. 

Active immobilized enzyme assay of cyanide in an enzyme thermistor Purification of human erythrocyte actin free from spectrin Active immobilized enzyme... 

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