Table 2.
Effect of selected constituents of pantethine and glutathione on the aggregation of ADH in the presence and absence of α crystallin.
| Standard (ADH + α) | OD(apparent)
|
|||
|---|---|---|---|---|
| 0.1 | 0.5 | 0.9 | Max = 1.0 | |
| +10 mM pantethine | 0.08 | 0.13 | 0.54 | 0.72 |
| +10 mM glutathione | 0.00 | 0.03 | 0.12 | 0.18 |
| +20 mM cystamine (-SS-) | 0.02 | 0.05 | 0.18 | 0.33 |
| +20 mM cysteamine (-SH-) | 0.07 | 0.44 | 0.85 | 0.94 |
| +20 mM cystine (-SS-) | 0.03 | 0.09 | 0.26 | 0.44 |
| +20 mM cysteine (-SH-) | 0.19 | 0.86 | 1.26 | 1.40 |
| +20 mM cysteamine (-SPO4) | 0.04 | 0.09 | 0.28 | 0.50 |
| +20 mM β-alanine | 0.07 | 0.38 | 0.62 | 0.66 |
| +20 mM glycine | 0.09 | 0.45 | 0.76 | 0.83 |
| +20 mM pantothenate | 0.08 | 0.51 | 1.00 | 1.10 |
| +20 mM glutamate | 0.14 | 0.62 | 1.03 | 1.14 |
| Standard (ADH alone) | ||||
| +20 mM pantethine | 0.02 | 0.03 | 0.59 | 0.97 |
| +20 mM glutathione | 0.01 | 0.02 | 0.07 | 0.26 |
| +20 mM GSH | 0.15 | 0.61 | 1.25 | 1.43 |
| +20 mM cysteine (-SH) | 1.61 | 1.72 | 1.89 | 1.89 |
| +20 mM cysteamine (-SH) | 0.10 | 0.50 | 0.93 | 1.03 |
| +20 mM cysteamine (-SPO4) | 0.01 | 0.02 | 0.34 | 0.84 |
We were most interested in the action of the test reagents at physiological temperatures, and further studies were conducted using ADH which undergoes aggregation and opacification at physiological temperatures of 37°C. The OD values for the standard solution of ADH plus α-crystallin without added reagents are listed in row 1 (standard). In the ADH plus α-crystallin assay, glutathione had a much stronger effect than the pantethine. The effects of the constituent molecules, glycine and β-alanine, glutamate and pantothenate, cystine and cystamine were analyzed. With the exception of the acids, glutamate and pantothenate, the constituent molecules of glutathione and pantethine enhanced the action of the α-crystallin. The strongest effects were observed with sulfur containing peptides. A study of selected -SH and -SS- forms of cystamine and cystine found that the oxidized -SS- forms were more protective than the reduced -SH forms and resulted in lower values for OD. The effects of selected reagents on aggregation of ADH without α-crystallin were evaluated. Both pantethine and glutathione inhibited aggregation of ADH alone although the effect of the glutathione was much stronger than the pantethine. The sulfhydryl reducing agents increased OD and aggregation of ADH alone. The OD was normalized as described.