Figure 2. Determination of disulfide... | Rockefeller University Press

Figure 2.

$Figure 2. Determination of disulfide cross-linking. (A) Scheme of the method, in which cleavage at the HRV-3C protease site in the S0–S1 loop of α produces an ∼15-kD N-terminal fragment and an ∼110-kD C-terminal fragment. Shown schematically are the species generated from different starting α forms after treatment with protease and then treatment with DTT. (B) Western blots illustrating cross-linking of Cys pairs and controls. The blots were developed with an antibody against a C-terminal epitope, reacting with both the 125-kD full-length α and with the 110-kD C-terminal fragment generated by HRV-3C protease cleavage. (i) Control showing that the mobility of pWT α is not affected either by DTT (lane 1) or by HRV-3C protease followed by DTT (lane 2). (ii) α with an HRV site is cleaved by HRV-3C protease and is unaffected by DTT. (iii) α in which the native pair, Cys14 and Cys141, is endogenously cross-linked, as shown by the preponderance of the 125-kD band after protease treatment (lane 1) and the loss of this band after protease and DTT (lane 2). In panels iv, v, and vi are shown the endogenous cross-linking of three additional pairs of Cys, one Cys in each case in S0 and one in S1 (iv), S2 (v), or S3–S4 (vi). At the bottom of each gel is the fraction of α cross-linked corrected for the efficiency of cleavage (see Materials and methods).$

Determination of disulfide cross-linking. (A) Scheme of the method, in which cleavage at the HRV-3C protease site in the S0–S1 loop of α produces an ∼15-kD N-terminal fragment and an ∼110-kD C-terminal fragment. Shown schematically are the species generated from different starting α forms after treatment with protease and then treatment with DTT. (B) Western blots illustrating cross-linking of Cys pairs and controls. The blots were developed with an antibody against a C-terminal epitope, reacting with both the 125-kD full-length α and with the 110-kD C-terminal fragment generated by HRV-3C protease cleavage. (i) Control showing that the mobility of pWT α is not affected either by DTT (lane 1) or by HRV-3C protease followed by DTT (lane 2). (ii) α with an HRV site is cleaved by HRV-3C protease and is unaffected by DTT. (iii) α in which the native pair, Cys14 and Cys141, is endogenously cross-linked, as shown by the preponderance of the 125-kD band after protease treatment (lane 1) and the loss of this band after protease and DTT (lane 2). In panels iv, v, and vi are shown the endogenous cross-linking of three additional pairs of Cys, one Cys in each case in S0 and one in S1 (iv), S2 (v), or S3–S4 (vi). At the bottom of each gel is the fraction of α cross-linked corrected for the efficiency of cleavage (see Materials and methods).