Abstract
In an effort to assess the influence that crystallization may have on protein conformations, optical absorption spectra of crystalline state hemoglobin derivatives have been examined. These spectra were obtained from photoacoustic spectra using a computer-assisted analysis. Comparisons of crystal and solution state hemoglobins using crystal minus solution state difference spectra indicate that the conformations of these proteins are similar in both states. Crystallization does not change the absorption properties of horse oxyhemoglobin or the cyanide and azide adducts of horse and human methemoglobin. Spectra of crystalline methemoglobins, which are prepared by ligand exchange in the crystalline state, are identical to the spectra of the final crystalline state adduct prepared without ligand exchange. Further, the allosteric effector, inositol hexaphosphate, causes the same spectral changes in solution and crystalline state hemoglobins. However, differences between crystal and solution state spectra of both fluoride and aquo methemoglobin are observed. These differences suggest that small but perhaps functionally significant changes in the heme regions of these derivatives accompany crystallization.
Highlights
From the Department of Biological Chemistry, College of Science and Engineering and School of Medicine, Wright State University, Dayton, Ohio 45435
Several observations suggest that crystallization may persorption spectraof crystalline state hemoglobin deriv- turb the functional properties of both hemoglobin and the atives havebeenexamined.Thesespectra were ob- structurally similar proteinm, yoglobin
Comparisons of crystal and solution even when crystal phase diffusion constraints are taken into state hemoglobins using crystal minus solution state account(Chance and Ravilly, 1966; Chance et al, 1966). etohldtiihxxoniofecyrnefshsheeearmedpemanoennrgetoocdeshgteeihelnnomsiunpobmtotseihngcaeaclotnohrrrcbaeatmrhiniysnenegissmtcdet,hywaiieacllthmlaahniirtneicoedhegieatnlshaobtnbbaaasdtiorotneertta.hh,pzSpeatisdpirrotceeeaeonpcatntiadedrpfrsdaoee.rurndooCmctpbftireycsayoraltisctfilgtioreaatynosnlslsdittozoahfafle--hotbsoIaronofinsnlfedaudMrtatisinrboloegeilnsdautstoasitnotpaodneatnrnetsemdutd(arriiMbetcsesersiaododskscpupiiieneaneretcmiintnotrhnowteeshftchrecooaarmplhLyeie,schmtMa1ocle9rbolsi7Cmpne9oOepH)c.aksbirAnteiarstddeotideqfcnriuastoritiemoerolenfasCaittilcmOvslryeyop,bsvotitlesnaoimgildttlaiiiheonnnnengedt spectra of the final crystalline state adduct prepared found by x-ray diffraction analysis
Summary
In the region of interest, 450 to 700 nm, calculated as described Fig. 4 shows crystal minus solution state difference spectra (Miniprint)is shown with the solution state absorption spectrum. Nm similarity is indicated by the absence of maxima or minima in predicted minus observed PA difference spectra (Fig. 2) or in PA-derived minus solution state absorption difference spectra (data not shown). Hemoglobin Derivatives That Are Spectrally Sensitive to Crystallization-The absorption spectra of several of the Hb derivatives examined were perturbed by crystallization ThepH 5.0 minus pH 7.0 difference spectrum of solution state horse Hb'I'F (dashed line, bottom) and the absorption spectrum of solution state human Hb"'F (top, dotted line and right hand anis) are shown
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