The pH dependence of tryptophan fluorescence of goat alpha-lactalbumin with particular reference to the effect of binding of an impurity from milk.

Two processes giving rise to changes in tryptophan fluorescence of goat alpha-lactalbumin below pH 7 have been studied. Both of these spectral transitions are strongly influenced by the presence of an impurity which binds to the protein if its contact at acid pH with the milk mother liquor is prolonged during its preparation. Transition II, the U leads to N conversion, follows the same course at both 2 and 25 degrees C, i.e. a minimum of three abnormally titrating groups are normalized during the conformational change at both temperatures. The difference in this conclusion from that made earlier (Kronman, M.J., Jeroszko, J. and Sage, G.W. (1972) Biochim. Biophys. Acta 285, 145-166) that groups are normalized at 25 and at 2 degrees C was found to be due to binding of the impurity to the protein used in the earlier study. Transition I, which we detect between pH 7 and 4.4 and which appears to be due to perturbation of tryptophans by vicinal ionizing groups, overlaps Transition II, the U leads to N conversion. However, the fluorescence above approx. 340 nm in the emission spectrum appears to be free of contributions from the former process and can be used in a valid analysis of the thermodynamics of the latter process.

Comparative fluorescence properties of bovine, goat, human and guinea pig alpha lactalbumin. Characterization of the environments of individual tryptophan residues in partially folded conformers.

alpha Lactalbumin exists as a partially folded conformer (U form) at acid pH. A second partially folded conformer (H form) is formed above 60 degrees. Comparison of the changes in tryptophan fluorescence which occur on forming U and H for the bovine, goat, human and guinea pig proteins, as well as analysis of fluorescence properties for the bovine protein and an N bromo succinimide derivative of this protein, have made it possible to determine which tryptophan residues give rise to such changes in fluorescence, and to draw a distinction between the molecular structure of the U and H forms of the protein. Trp 28 and 109 in the native state transfer their excitation energy to trp 63 whose fluorescence is quenched by a pair of vicinal disulfide bridges. This process persists in the U form of the protein, but is absent in the H conformer. Most of the change in fluorescence seen in the N in equilibrium with U conversion is due to increase in yield of trp 28, while the changes in fluorescence occurring on formation of the H form are due to exposure of trp 63 and elimination of its quenching and/or excited state transfer from 28 to 109.

Direct determination of total serum cholesterol by use of double-wavelength spectrophotometry.

We describe a simple, accurate method for direct determination of total cholesterol in serum. Systematic investigation of a previously described modified Liebermann-Burchard reagent has indicated the necessity of accounting for both bilirubin interference and decreased specificity owing to exothermia. Double-wavelength spectrophotometry was used to optically null out bilirubin as an interfering factor, whereas adding serum to the cold reagent increases its specificity for the cholesterol color reaction. Comparison of 106 cholesterol values with those obtained by the procedure of Abell et al. [J. Biol. Chem. 195, 357 (1952)] yielded a correlation coefficient greater than 0.99; our inter-run coefficient of variation of polled laboratory serum was 1.7%.