This paper describes several experimental and computational methods which are currently used in the structural analysis of metal-containing macromolecules. A specific family of proteolytic enzymes which contain a zinc cation in the active site was selected to demonstrate these methods. A range of studies using one example from this family of enzymes is described which serves to clarify the role of the metal in the overall protein structure and in the local conformation of the active site in the native enzyme, the metal-deficient enzyme, and the metal-substituted enzyme and in complexes of the enzyme with various chemical analogues. The main experimental method described is X-ray crystallography, while computational methods for the examination of surface interactions and electrostatic potential effects are described briefly to complement the structural conclusions. The various experimental and computational results are then assembled in order to draw general conclusions on the structure-function relationships of metalloproteins and in particular the role of the metal in metal-containing proteolytic enzymes. The results of these studies implicate the zinc ion in the binding and catalytic activation of the substrate and stabilization of the tetrahedral reaction intermediate. It appears that in this family of enzymes a divalent metal cation is important for the required catalytic arrangement of functional groups in the active site, especially the metal ligands. However, once an appropriate metal ion is coordinated, there is practically no effect of the particular metal ion bound on either the overall three dimensional structure of the enzyme or the local detailed structure of its active site.

Amino Acid Sequence↗

PubMedDate not supplied

Ultrasonic detection of red cell aggregation immediately preceding blood clotting.

High-resolution ultrasonic imaging of circulating blood was used to study the relation between red cell aggregation and blood clotting in vitro. A reversible increase in echogenicity produced by red cell aggregation occurred in moving heparinized blood as shear rate was decreased. We induced clotting of the heparinized blood by administration of protamine. At both low (1.6 sec-1) and moderate (22.6 sec-1) mean shear rates, transient homogenous increased echogenicity indicative of red cell aggregation preceded blood clotting. In separate experiments, we established that protamine can cause increased echogenicity due to red cell aggregation which can be reversed by adding heparin to circulating suspended red cells in the absence of clotting factors. Presumably, these effects of protamine and heparin are due to electrostatic bonding involving red cell surfaces. We conclude from these studies that red cell aggregation precedes clotting of heparinized blood by protamine at low and moderate shear rates.

Blood Coagulation↗