Evaluation of the thermal stability of clinically relevant enzymes at 37 degrees C.

The thermal stability at 37 degrees C of several clinically relevant enzymes and isoenzymes was assessed by measuring changes in enzyme activity as a function of time under incubation and reaction conditions. Selwyn plots were used in the reaction-condition assessments. Except for CK-1 (BB), all the enzymes investigated are stable enough at 37 degrees C to permit assay. These enzymes were LDH-1, LDH-5, s-AspAT, m-AspAT, apo-s-AspAT, apo-m-AspAT, ALP-liver, ALP-bone, ALP-intestine, ALT, apo-ALT, CK-2, and CK-3. CK-1 is stable at 37 degrees C under assay conditions but not under incubation conditions. We specifically avoided using Arrhenius plots to evaluate thermal stability and point out pitfalls inherent in their indiscriminate use.

Effects of glucose and magnesium ion on the quenching of yeast hexokinase fluorescence by acrylamide.

To probe the effects of the substrate, glucose, and the cofactor, Mg2+, on the structure of hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1), titrations of the tryptophan fluorescence of yeast hexokinase isozyme P-II(B) were performed. Acrylamide was used as a quenching titrant in the absence and in the presence of glucose and Mg2+ singly and together at pH 5.5 and 8.3 at 20 degrees C. The four tryptophan residues of the monomeric subunit of yeast hexokinase may be classified as two surface residues, one being highly accessible to dissolved I- and one with restricted accessibility to I-, one glucose-quenchable residue in the cleft, and one buried (Kramp, D.C. and Feldman, I. (1978) Biochim. Biophys. Acta 537, 406--416). The acrylamide data were analyzed by least-squares computer analysis for quenching constants and fractional fluorescence values of the tryptophan residues. The quenching constants measure the accessibilities of the residues to the quencher, while the fractional fluorescences are related to the microenvironments of the fluorophores. At each pH value, glucose altered the quenching constants, but not the fractional fluorescence, of the tryptophan residues. Mg2+ greatly accentuated at this glucose effect, especially for the surface residue near the cleft opening. Comparison of acrylamide- and I-quenching data shows that this particular residue has a positively charged microenvironment. A pH change from 5.5 to 8.3 increased the acylamide-accessibility of the cleft tryptophan but did not seem to influence accessibility of the surface residues or the buried residue significantly, thus strengthening our previous conclusion that the cleft opening is small enough at pH 5.5 to partially restrict entrance of organic molecules and negative ions. However, with saturating glucose present there was a pH effect on the surface residue accessibility. Titrations in 55 vol.% glycerol suggest the presence of transient channels (not just holes) in the hexokinase structure, which allows penetration of the protein by solution. Consequently, the buried tryptophan residue is quenched more strongly by dissolved acrylamide than is attributable to diffusion of quencher through the protein matrix.

Effects of free magnesium and alkali ions on the conformation and glucose-binding strength of yeast hexokinase isozymes.

Titrations of the tryptophan fluorescence of yeast hexokinase (ATP:D-hexose 6-phosphortransferase, EC 2.7.1.1.) isozymes P-I (A) and P-II (B) were performed with Mg2+, Li+, Na+ and K+ as titrant in absence and in presence of glucose, and vice versa, at pH 8.3 and 5.5 at 20 degrees C. Mg2+ quenches the fluorescence of surface tryptophan primarily and does so by producing a conformational change which alters the microenvironment of the tryptophan. For both isozymes Mg2+ exerts a specific ion effect, i.e. significantly larger than the ionic strength (I) effect, which enhances the glucose quenching by causing a conformation change which increases the glucose-binding constant. For the P-I isozyme glucose binding exhibits positive cooperativity at both pH 8.3 and 5.5 when the ionic strength (I) is low, i.e. significantly larger than the ionic strength (I) effect, which enhances the glucose quenching by causing a conformation change which increases the glucose-binding constant. For the P-I isozyme glucose binding exhibits positive sooperativity at both pH 8.3 and 5.5 when the ionic strength (I) is low, i.e. 0.04 or less, regardless of which of the above four cations is present. For P-II, however, glucose binding is non-cooperative at pH 8.3 regardless of I or the cation species and at pH 5.5 and low I with K+ or Mg2+ as the predominant cation present, but there is apparent negative cooperativity at pH 5.5 and low I when Na+ or Li+ predominates. These results are discussed in terms of known structural characteristics of the isozymes.