Anisotropy to Study Proteins

Strambini and Galley have used tryptophan anisotropy to measure the rotation of proteins in glassy solvents as a function of temperature. They found that the anisotropy of tryptophan phosphorescence reflected the size of globular proteins in glycerol buffer in the temperature range -90 to -70°C.(84 85) Tryptophan phosphorescence of erythrocyte ghosts depolarized discontinuously as a function of temperature. These authors interpreted the complex temperature dependence to indicate protein-protein interactions in the membrane. 

The rotational mobility of human low-density (LDL) and very-low-density (VLDL) lipoproteins was studied as a function of viscosity and temperature in the range of —90 to — 50°C.(86)The rotational behavior for LDL is represented by a single correlation time, consistent with the overall rotation of a spherical rigid particle as the source of the phosphorescence depolarization. For VLDL, internal peptide motions dominate the depolarization profile. 

Berger and Vanderkooi(88) studied the depolarization of tryptophan from tobacco mosaic virus. The major subunit of the coat protein contains three tryptophans. The phosphorescence decay is non-single-exponential. At 22°C the lifetime of the long component decays with a time constant of 22 ms, and at 3°C the lifetime is 61 ms. The anisotropy decay is clearly not singleexponential and was consistent with the known geometry of the virus. 

The long-lived phosphorescence of the tryptophan in alkaline phosphatase is unusual. Horie and Vanderkooi examined whether its phosphorescence could be detected in E. coli strains which are rich in alkaline phosphatase.(89) They observed phosphorescence at 20°C with a lifetime of 1.3 s, which is comparable to the lifetime of purified alkaline phosphatase (1.4 s). Long-lived luminescence was not observed from strains deficient in alkaline phosphatase. The temperature dependence of tryptophan phosphorescence in the living cells was slightly different from that for the purified enzyme, indicating an environmental effect. 

Mazhul et alP have reported that long-lived luminescence could be detected in intact human erythrocytes and white blood cells at ambient temperature. They have shown by emission spectra and pH dependency that this emission arises from tryptophan. The emission was not singleexponential, suggesting that more than one population of tryptophan emitted. Identification of the emitting species has not yet been conclusively made, but the white blood cell protein content is about 10% actin, a protein known to phosphorescence.(91) 

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