The structure of haemoglobin - IV. Sign determination by the isomorphous replacement method

Abstract

Native horse haemoglobin contains free sulphydryl groups and forms crystalline compounds with para -mercuribenzoate groups and with silver ions. Crystals in which two of the four available SH groups are so combined are exactly isomorphous with normal monoclinic methaemoglobin, but exhibit significant changes in the intensities of many reflexions. The changes in F ( h 0 l ) were used to determine the x and z parameters of the pair of heavy atoms attached to each haemoglobin molecule; this was done both for the normal wet lattice and for one of the acid-expanded lattices. The positions of the heavy atoms proved to be slightly different in each case, giving rise to three sets of diffraction fringes, each set making measurable contributions in different areas of the reciprocal net. In each case the isomorphous substitution allowed the signs of just over two-thirds of the reflexions to be found with certainty. Between them the three sets of diffraction fringes determined the signs over the entire area of the h 0 l plane so far investigated. These signs were then superimposed on the waves of the transform described in previous papers of this series. All the sign relations established by the transform method were confirmed and the remaining uncertainties cleared up. Comparison of the transform with the three sets of isomorphous replacement results allowed the consistency of the signs to be rigorously checked; not a single inconsistent sign was found. In the normal wet lattice the mercury and the silver compounds between them allowed the signs of 87 out of 94 reflexions to be found with certainty. This suggests that the isomorphous replacement method may offer a way of finding the phases in protein crystals even when practical difficulties preclude the use of the transform method.