Authentic acid blood mordanted the madder-dyed Shroud of Turin pinkish red before image formation – Jesus was dead © A.A.M. van der Hoeven – September 22, 2014 www.JesusKing.info – https://delftschoolofdesign.academia.edu/AdrieVdHoeven corrected February 27, 2015 ABST RACT The chemical and physical properties of the Shroud of Turin, both incompatible with almost all properties of the Shroud and of the non-image background, the image areas, the pinkish red bloodstains. bloodstains and the separate serum, especially their absorbance, A realistic mechanism of formation of the separate serum coated fluorescence and FTIR spectra, are described and shown to be fibers and different types of bloodstains – moist drying-clot evidence for the presence of a pinkish red heme-madder imprints especially on the ventral half of the Shroud, blood complex, formed by the chemical interaction of authentic acid rivulets across the dorsal half of the Shroud, and later imprints of postmortem blood with yellow madder dye on the cloth before fybrinolysed scourge wound clots on both halves of the Shroud – image formation. Besides the many normal blood characteristics, is described and complies with the events that led to the death the lack of potassium of the red bloodstains indicates that the and burial of Jesus, as recounted in the Gospels: scourging and blood was hyperkalemic: that potassium had moved out of the crowning with thorns before midday, carrying the cross uphill, red blood cells into the plasma, which inevitably occurs in crucifixion and then death in the middle of the afternoon, a acidemic blood. The absorbance spectra of the bloodstains stabbing in the side, and then the deposition, wrapping in linen indeed show the typical charge transfer band of acid blood at 630 and transferral to the tomb – allowing the re-opening of nm. The lack of red blood cells, the lack of a large Soret band in crucifixion and head wounds, the formation by wet bloodstained the bloodstains’ absorbance spectra (at ca. 400 nm), and the hands of bloodsmears outside the body contours and a peculiar pinkish-red color of the bloodstains indicate that, after a bloodsmear possibly on the edge of an oval first-century Jewish draining away of the potassium-rich plasma/serum, the red blood ornament (the petalon of a Sanhedrin member such as Joseph of cells hemolysed (broke open) and their hemoglobin denatured Arimathea) that was laid on the chest of the body, and the further to acid heme dimers – which only have a small Soret running of irregular blood rivulets across the dorsal half of the band – and that these acid heme dimers mordanted the yellow waterresistant Shroud on a shoulder-carried water-absorbing madder dye on the Shroud pinkish red. The FTIR spectra of wooden burial bier –, and finally the laying down of the body (plus Shroud bloodglobs are similar to those of madder lake. Also the bier) in the tomb at the end of the afternoon. The strenuous reddish color of the bloodstain fluorescence corresponds to the exercise and the crucifixion of several hours would have implied reddish fluorescence of madder lake, and the shape of the mean severe cramping, respiratory failure, dehydration and thus severe UV-vis reflectance spectrum of four bloodstains is compatible antemortem acidemia that inevitably increased after death and with a combination of madder dye, madder lake and heme. then apparently started a hyperfibrinolysis process (a The presence of a thin coating of (acid) retrograded starch and remoistening of blood clots on the wounds, from within the body), acid madder dye on the superficial fibers of the Shroud is as is evidenced by the pattern of unsmeared dumbbell-shaped evidenced by the strippable coating in all Shroud areas, the imprints of scourge wound clots. Hyperfibrinolysis only starts up erasal of superficial scorches and background color by hot at an acid blood pH < 6.8, which blood pH is incompatible with firefighting water, leaving blue fluorescent areas, and the similar life but compatible with the absorbance spectra of the Shroud’s FTIR spectra of the main Shroud’s background and those of the bloodstains. madder-and-starch-containing radiocarbon-dating corner, As the image fibers have lost their surface of easily oxidizable compatible with those of linen, starch and madder dye. Also the madder dye in the oxidative image formation process, evidenced visible pinkish hue, the acidichromism, reflectance spectra, by the image color and fluorescence, and as the bloodstains in fluorescence spectra, and yellow-green fluorescence of the image areas are all the same pinkish-red color and their background, the weaker image fluorescence, the blue-fluorescent reflectance spectra have the same shape, independent of the ‘no-print’ areas, and the microscopic observations are evidence concentration of image fibers that would have been present in the for the presence of madder dye on the Shroud. Madder dye is bloodstain locations, the blood must have formed pinkish heme- soluble in xylene and in toluene, the solvents that were used to madder lake before the start of image formation. This, and the wash the adhesive from Shroud fibers stuck to sticky tape realistic appearance of the bloodstains and the presence of samples of the main Shroud. This may explain why no positive separate serum, as evidenced by FTIR spectra, precludes that wet-chemistry evidence of dye on fibers from the main Shroud the bloodstains were painted on a pre-existent, e.g. artificial, was reported, but, much later, madder was easily found by wet body image. Some well known red-blood hypotheses, such as chemistry on whole thread samples from the radiocarbon-dating those based on an ultra-high bilirubin level in the blood, UV- corner, which already has been shown to be no repair. To this irradiation of the blood, a Saponaria soap residue on the cloth, a can be added that FTIR spectroscopy found pectin but no protein nitric oxide ligand in a blood paint, and a red ochre paint are of gum Arabic on both the main Shroud and radiocarbon-dating analysed and shown to be unable to explain all the Shroud’s corner. The starch-and-madder coating served as many agents bloodstain characteristics consistently. Experiments showed that in the Shroud’s history. It was probably originally applied as a blood can form pinkish stains on starched and madder-dyed fluoresceing fabric brightner for an authentic Jewish temple linen. mantle, and it was a waterresistant filter separating the That there is a pinkish red bloodstain on the image of probably a potassium-poor red blood cells and the potassium-rich serum of first-century Jewish petalon, fits the high-priestly burial of Jesus acid hyperkalemic postmortem blood, a saponin layer hemolysing Christ in the Shroud of Turin by Joseph of Arimathea. That the the red blood cells, a denaturing and adsorbing substrate for Shroud shows no signs of putrefaction of the dead body, which pinkish heme-madder lake formation, an acid catalyst and easily would have started about 40 hours after death, and that the oxidizable substrate for acid oxidation in image formation in only dumbbell-shaped scourge marks on both halves of the Shroud this ultra-thin and strippable fiber coating, an acid coating that are not smeared, means that the dead body and its image- allowed the formation of pure iron oxide particles on the outer bearing Shroud separated in an extremely delicate way that did edges of the large waterstains from cellulose-bound iron, and not smear the moist fibrinolysed blood clots on the scourge finally a potent preservative for serum and heme and cloth. A wounds, which were still dry when the dead body was wrapped coating with Saponaria (soapweed) residues is shown to be into the Shroud. This fits the bodily Resurrection of Jesus Christ.• 1 Table of Contents 1. INTRODUCTION ............................................................................................................................. 4 1.1. Normal blood features ................................................................................................................. 4 1.2. Special features of the bloodstains .............................................................................................. 5 1.3. Analysis in this paper .................................................................................................................. 5 2. COHERENCE OF SPECIAL BLOOD FEATURES ........................................................................ 6 2.1. Red color but no Soret band ........................................................................................................ 6 2.1.1. Acid heme dimers ................................................................................................................ 7 2.1.2. Heme-madder lake ............................................................................................................. 23 2.1.3. Blood before image ............................................................................................................ 62 2.2. Separate serum - UV-fluorescence halo on wrist ..................................................................... 64 2.2.1. Identification of separate plasma/serum ............................................................................ 64 2.2.2. No fluorescent “serum” scratches but dark images of stripes ............................................ 71 2.2.3. Some “serum” margins possibly a tenting effect around other (parts of) bloodmarks ...... 72 2.3. No potassium signal in three X-ray fluorescence spectra of bloodstains ................................. 74 2.3.1. Postmortem blood is hyperkalemic .................................................................................... 74 2.3.2. Vertical serum draining ...................................................................................................... 76 2.3.3. Horizontally and vertically imprinted serum halos ............................................................ 78 2.3.4. Filter effect ......................................................................................................................... 82 2.4. Few cells – hemolysate stains ................................................................................................... 83 2.4.1. Separate serum not red ....................................................................................................... 85 2.4.2. Hemolysis mechanisms ...................................................................................................... 85 2.5. Hydroxyproline in red particles on Zina-thread ........................................................................ 91 2.6. High Na and Cl levels on reverse side ...................................................................................... 92 3. SURVIVAL OF CLOTH, BLOOD AND SERUM – PRESERVATIVE COATING .................... 94 3.1. Myrrh and aloes – antibacterial and antifungal ......................................................................... 94 3.2. Saponaria – antibacterial and antioxidant ................................................................................. 95 3.3. Madder – antimicrobic, antifungal, insecticidal, antioxidant ................................................... 96 3.4. Leech saliva antibiotics ............................................................................................................. 97 3.5. Mordant protects madder lake from degradation ...................................................................... 97 4. MADDER ON STARCH COATING .............................................................................................. 98 4.1. Starch ...................................................................................................................................... 100 4.1.1. Strippable sealing film ..................................................................................................... 100 4.1.2. Hot water washed out starch – blue fluorescence ............................................................ 103 4.1.3. FTIR spectra of Raes samples are similar to FTIR spectra of main Shroud non-image fibers and of linen ........................................................................................................... 105 4.2. Madder dye ............................................................................................................................. 139 4.2.1. Visible color and wet acid-base chemistry ...................................................................... 139 4.2.2. Reflectance curves of clear areas - raw and absolute ...................................................... 148 4.2.3. Raw fluorescence scan background ................................................................................. 151 4.2.4. Fluorescence photography ............................................................................................... 155 4.2.5. Image fluorescence .......................................................................................................... 163 4.2.6. SEM-EDS analysis – smooth organic coating embedding particles ................................ 166 4.2.7. Microscopy - Red aluminum lake particles ..................................................................... 167 4.2.8. Pyrolysis/Mass Spectrometry ........................................................................................... 172 4.3. Not pectin or microbial bioplastic coating .............................................................................. 173 4.4. Not Saponaria .......................................................................................................................... 174 4.4.1. Acidichromism – not Saponaria ....................................................................................... 175 4.4.2. Fluorescence – not quite Saponaria ................................................................................. 176 2 4.4.3. UV-vis absorbance – not Saponaria ................................................................................. 178 4.4.4. Sugars – no Saponaria evidence ...................................................................................... 178 4.4.5. Solubility – not Saponaria ................................................................................................ 180 4.4.6. Color with iodine – not Saponaria ................................................................................... 181 4.4.7. Effect on chelated iron – not Saponaria ........................................................................... 181 4.4.8. Effect on image formation – not Saponaria ..................................................................... 182 4.4.9. Lake colour with Al3+ and Ca2+ – not Saponaria ............................................................. 182 4.4.10. Heme-complex colour – not Saponaria .......................................................................... 182 4.4.11. Relative reflectance of bloodstains – not Saponaria ...................................................... 184 5. FORMATION MECHANISMS .................................................................................................... 186 5.1. Post-mortem heme dimer formation – further acidification, hemolysis and heme adsorption by dyed cloth – powder formation and abrasion .................................................................... 186 5.2. Blood drying on the body ....................................................................................................... 192 5.3. Rivulets running across the Shroud ........................................................................................ 194 5.4. Pools of wet blood – brown bloodstains ................................................................................. 196 5.5. Scourge marks ......................................................................................................................... 196 5.5.1. Very faint – not dense – not chemically tested – no spectra ............................................ 196 5.5.2. No fluorescent serum scratches or serum borders ........................................................... 200 5.5.3. Only dorsal scourge marks on reverse side ...................................................................... 200 5.5.4. Hyperfibrinolysis caused pink imprints but no smears before image formation ............. 200 5.5.5. Other ways of scourge mark transfer ............................................................................... 206 5.6. Blood smears from hands of buriers ....................................................................................... 208 6. OTHER RED COLOR HYPOTHESES ........................................................................................ 209 6.1. Authentic blood ....................................................................................................................... 209 6.1.1. Blood of a living, crucified person .................................................................................. 209 6.1.2. Bilirubin ........................................................................................................................... 209 6.1.3. Prior UV-irradiation ......................................................................................................... 215 6.1.4. CO-ligand from carbon monoxide gas ............................................................................. 216 6.1.5. Saponaria-treated cloth .................................................................................................... 216 6.2. Painted-on bloodstains ............................................................................................................ 217 6.2.1. ‘Cured’ blood paint – NO or CO ..................................................................................... 217 6.2.2. Iron oxide particles in protein binder ............................................................................... 221 6.2.3. Iron-madder lake .............................................................................................................. 221 6.2.4. Acid blood ........................................................................................................................ 222 6.3. Survey red color hypotheses ................................................................................................... 222 7. BLOOD ON THE PETALON - NOT ON THE BEARD ............................................................. 224 8. CONCLUSION .............................................................................................................................. 230 9. ACKNOWLEDGEMENTS ........................................................................................................... 232 Corrected errors ............................................................................................................................. 232 Bibliography .................................................................................................................................. 233 3 1. INTRODUCTION The Shroud of Turin is a 4.4 x 1.1 m linen cloth bearing the ventral and dorsal image of a man and apparent bloodstains in the areas of the hands, feet, side, head, and small of back, and also burn holes and scorch marks and waterstains (fig. 1). It is kept in the cathedral of Turin, Italy, and believed by many to be the burial cloth of Jesus Christ. Fig. 1. The Shroud of Turin1 1.1. Normal blood features It has been reported that the apparent bloodstains of the Shroud show a number of normal blood and serum features, as evidenced by several techniques, such as the following: - red blood cells (29 of 500 particles) – microscopy, SEM, X-ray microfluorescence – Lucotte2 - a human red blood corpuscle with the characteristic elements of blood – SEM and X-ray microspectrometry – Baima Bollone3 - some cell-like structures, iron, antigen B, adult hemoglobin – Wright’s technique, Prussian blue test, immunohisto-chemical tests – Garza-Valdez4 - hemoglobin – Raman analysis – Baraldi5 - hemochromagen-like color and cyanmethemoglobin type color producible – Heller and Adler6 - heme/porphyrins – Dotzauer and Keding method, Teichman crystals and hematine chlorohydrate producible – Baima Bollone7 - porphyrin fluorescence producible – after displacement of iron by treatment with hydrazine and formic acid vapor – Heller and Adler8 - iron concentration consistent with bloodstains – X-ray fluorescence – Morris, Schwalbe and London9 - the elements Mg, Al, Si, S, Cl, K, Ca and Fe – EDS - Baima Bollone10 - proteins – protease test and fluorescamine test– Heller and Adler11 - shiney honey yellow colored fibers, a deeper color than the image – microscopy – Schwalbe and Rogers12 1 Unofficial photo http://wpmedia.life.nationalpost.com/2013/03/shroud-of-turin.jpg from http://life.nationalpost.com/2013/03/29/shroud-of-turin-not-a-medieval-forgery-researchers-say/ ; see also https://www.shroud.com/examine.htm and http://www.sindonology.org/shroudScope/shroudScope.shtml 2 Lucotte, Optical and chemical…, 2012, p. 2547 3 Baima Bollone, The Forensic Characteristics…, Proceedings Turin 2000, p. 213 4 Garza Valdez, The DNA of GOD?, 2001 p. 112-113 5 in Fanti and Gaeta, Il mistero della Sindone, 2013, p. 180 6 Heller and Adler, A Chemical…, 1981, TOM 39 7 Baima Bollone, The Forensic Characteristics…, Proceedings Turin 2000, p. 212 8 Heller and Adler, Blood on the Shroud of Turin…, 1980, The Orphaned Manuscript (TOM) p. 31 9 Morris, Schwalbe and London, X-Ray Fluorescence…, 1980, p. 40-42 10 Baima Bollone, The Forensic Characteristics…, Proceedings Turin 2000, p. 210 (plot of his EDS spectrum is online in Kearse and Heimburger, The Shroud Blood Science…, 2013, p. 4) 11 Heller and Adler, A Chemical…TOM 40-41 and table 4; Adler, The Nature of… , 1999, TOM 106-107 12 Schwalbe and Rogers, Physics and Chemistry…, 1982, p. 37 4 - albumin – Bromcresol Green test – Heller and Adler13 - primate immunoglobin – immuno-chemical tests – Heller and Adler14 - blue fluoresceing parts (of serum?) of vacuumed loose red particles - Fanti15 - human immunoglobin and human serum – direct and indirect immunological method – Baima Bollone16 - erythrocyte antigens A and B – immunohistiochemical technique conjugating antisera with raphanin peroxidase – Baima Bollone17 - human X- and Y-chromosomes – advanced DNA technologies – Tryon18 - human blood – Canale 199519 - realistic appearance: the borders of the forehead stains and of stains on the hair image are more coloured than their centres – Baima Bollone20; this is typical for blood clots formed on the skin – Barbet21. 1.2. Special features of the bloodstains Remarkable features of the apparent bloodstains on the Turin Shroud are: - the presence of both many pinkish red bloodstains and a few brown bloodstains (see 5.4) - the absence of a detectable Soret absorbance band (see 2.1) - the absence of a detectable XRF potassium signal from bloodstains on the Shroud (see 2.3) - few, almost no, cells in the bloodstain surfaces (see 2.4) - the resistance of the bloodstains and serum haloes to microbial attacks of ≥650 years (see 3) - the presence of blood stains that were not in a body-sheet contact zone, if the Shroud was only loosely draped over a supine body that was lying on one half of the Shroud (see 5.6) - the presence of a pattern of many pinkish red stains in a well-defined dumbbell form (see 5.5.4) - the presence of hydroxyproline in red spots on the ‘Zina-thread’ from a blood area on a heel image (see 2.5) - relatively high Na and Cl levels in organic red particles vacuumed from the reverse side of the Shroud (see 2.6) 1.3. Analysis in this paper In this paper a consistent explanation of both normal and special features is presented (chapter 2 and 3 and 5), which is based on already published evidence and on results of new experiments, and which is also in congruence and evidenced by the properties of the background and image of the Shroud (chapter 4), and in congruence with the earlier proposed identification of the Shroud as a madder- dyed Jewish temple mantle with Pharisaic border without corner repair.22 Other hypotheses regarding the redness of the bloodstains are also analysed, but dismissed as incongruent with the Shroud’s properties (chapter 6). Also new evidence for the Shroud’s presence in an authentic Jewish burial procession for the burial of Jesus is presented (chapter 7). 13 Heller and Adler, A Chemical…, 1981,TOM 40, 36, 50 table 2 14 Heller, Report on…, 1983, p. 188 15 Fanti, Statistical analysis…, 2008, fig. 10, 16 Baima Bollone, The Forensic Characteristics…, Proceedings Turin 2000, p. 212; discussed in Kearse, Blood on the Shroud of Turin: An Immunological Review, 2012 17 Baima Bollone, The Forensic Characteristics…, Proceedings Turin 2000, p. 212 18 in Wilson, Science Fiction to Science Fact?, 1996 19 “the presence of human blood was subsequently confirmed by Canale in 1995 before conducting DNA research on some threads I gave him”, Baima Bollone, The Forensic Characteristics…, Proceedings Turin 2000, p. 214 20 Proceedings Turin 2000, p. 215-216 21 in Brillante, Fanti and Marinelli, Bloodstain characterisics…, 2002, p. 5, 8 referring to Barbet 22 Hoeven, The seam and missing corners…, 2011-2013; Hoeven, Internal selvedge…, 2012 5 2. COHERENCE OF SPECIAL BLOOD FEATURES 2.1. Red color but no Soret band Adler reported on the Shroud blood: “It all looks perfectly acceptable for blood except for one thing – it is too red for blood that is supposed to be some 600 to 2000 years old. Everyone knows that blood changes color when exposed to the air; it changes to a methemoglobin which gives it a brown color. So we need to explain why the centuries-old blood on the Shroud is still so red.”23 Baima Bollone reported the same problem: “Another open question is that of the colour of the bloodstains on the Shroud. People have always been astonished by their bright red colour while it is known that bloodstains become brown with the gradual passing of time.”24 For an impression of the color, see fig. 2.1, from Shroud Scope. The following analysis was given by biochemist Berry: “Scientifically speaking, one should ask if the stains are consistent with their being extensively- degraded human blood (EDHB). But then one has to agree on what the markers are for EDHB. What would be the state of the original haemoglobin in the red blood cells, for example? Intact haemoglobin – improbable, even as (oxidized) methaemoglobin with iron(III), or free haems, i.e. non-proteinaceous iron-complexed porphyrins (more likely, but with what ligand? Jackson’s CO? Pull the other one) or iron-free porphyrins (even more likely), end-stage iron oxide, Fe O or Fe O (possibly).”25 2 3 3 4 23 Adler, The origin and nature…, 1986, TOM 60 24 Baima Bollone, The Forensic Characteristics…, Proceedings Turin 2000, p. 217 25 Berry, Shroudie-Alert: Day 9. Is it real human blood on the Shroud? A reply to Richard Savage, aka Jabba., “Casting a critical eye on that Shroud of Turin”, Feb 27, 2013, http://shroudofturinwithoutallthehype.wordpress.com/2013/02/27/shoudie-alert-day-9/ (line breaks added here for easier reading) 6 Fig. 2.1. Blood in area of the soles of the feet, screenshot of Shroud Scope, Durante 200226 2.1.1. Acid heme dimers 2.1.1.1. Acid heme Three kinds of original UV-Vis absorbance data from Shroud blood material are available: 1) a “brownish red translucent crystal” (“named biltong”) that in transmission showed a Soret absorption band at 405-410 nm and probably more bands – as it was identified as an acid methemoglobin crystal – but these are not specified or shown;27 2) a “garnet red” stained fibril that in transmission showed an absorption band at ca. 450 nm, with a shoulder at ca. 530 nm (Adler mentions 520 nm as one of “the observed peaks”28), but, according to Adler, these may have been shifted and distorted due to a high degree of scattering29 (fig. 2.2.A); 26 Latendresse, who publishes www.sindonology.org, also offers Shroud Scope (http://www.sindonology.org/shroudScope/shroudScope.shtml ) with the option of a permalink to any screenshot, e.g. this one http://www.sindonology.org/shroudScope/shroudScope.shtml?zl=7&image=3&lon=1947.0&lat=1015.0 . Image used with permission from Latendresse. 27 Heller and Adler, Blood on the Shroud…, 1980, TOM 31; Heller, in his book, described the appearance of a clear Soret band at 405-410 nm in the spectrum from the crystalline Shroud sample, measured in transmission, and identified as acid methemoglobin: Heller, Report on…, 1983, p. 144-147, read online at http://books.google.fr/books?ei=dNPnUbiBLoL_PKergcAH&hl=fr&id=o9glK7LcmHUC&dq=heller+report+on+the+shr oud+of+turin&q=acid+methemoglobin and http://books.google.fr/books?ei=dNPnUbiBLoL_PKergcAH&hl=fr&id=o9glK7LcmHUC&dq=heller+report+on+the+shr oud+of+turin&q=hit+400 28 Adler, Updating recent…, 1996, TOM 84 (see quote in 6.1.2.3) 7 3) pinkish red Shroud bloodstains that in relative reflectance show no distinguishable Soret absorption but a broad absorption band from ca. 340-525 nm and a clear narrow absorption band at 630 nm (fig. 2.3.), although, according to Adler, this spectrum is also distorted due to anomalous dispersion.30 Fig. 2.2. Absorbance spectra: A of “a brownish red stained fibril from one of the blood areas of the Shroud”; “B, transmission spectrum obtained by transformation of the reflection spectrum of the blood areas of the Shroud” ©OSA 31 Fig. 2.3. Mean relative reflectance values of four big bloodstains on the Shroud ©OSA 32 29 The investigated Shroud fibers are called “a single microfiber … garnet red” (Heller, Report on 126), and “seven microfibers with red stains on part of their length” (Heller, Report on…, 1983, 128) and “brownish red” (Heller and Adler, Blood on the Shroud…, 1980, TOM 30 and its fig.2 on TOM 31) by Heller and Adler, who saw these samples through the microscope, probably in transmitted light. See their article pages (TOM 30-31) with a plot of one of the transmission spectra showing a strong absorption band at 450 nm in fig. 2.2 A or through this link: http://books.google.nl/books?id=J2jBnDN3VxMC&pg=PA30&lpg=PA30&dq=spectral+data+of+the+Shroud+blood&source=bl&ots=zenDjCt69- &sig=WmPZiDBpkxQp61QSspVy4ZjVddk&hl=en&sa=X&ei=F3djUc- uOYmi8gTL1oCIBw&redir_esc=y#v=onepage&q=spectral%20data%20of%20the%20Shroud%20blood&f=false 30 Heller and Adler, Blood on the Shroud…, 1980, TOM 30-31 31 Used from J.H. Heller and A.D. Adler, Blood on the Shroud of Turin, Applied Optics, 19 (16) 1980, pp. 2742-2744, http://dx.doi.org/10.1364/AO.19.002742, fig. 2, with permission from the Optical Society of America. (online at A.D. Adler, The Orphaned Manuscript (TOM) p. 31, http://books.google.nl/books?id=J2jBnDN3VxMC&pg=PA30&lpg=PA30&dq=spectral+data+of+the+Shroud+blood&source=bl&ots=zenDjCt69- &sig=WmPZiDBpkxQp61QSspVy4ZjVddk&hl=en&sa=X&ei=F3djUc- uOYmi8gTL1oCIBw&redir_esc=y#v=onepage&q=spectral%20data%20of%20the%20Shroud%20blood&f=false 8 A characteristic of acid methemoglobin A (human methemoglobin = iron(III) protoporhyrin IX bound to globin, a protein) is the absorption peak at ca. 630 nm, the so-called charge transfer band.33 It gradually disappears when the acid methemoglobin is neutralized or gets in alkaline conditions, giving a growing ca. 575 nm absorption peak in return: one of the Q twin bands α and β.34 In acid methemoglobin there is an actual peak at 630 nm at pH 1 to at least 5 (fig. 2.4.), and a peak at 500 nm, and the Soret peak is at 405/406 nm;35 “an optical spectrum distinguished by relative intense maxima near 496 and 622 nm, which is a signature of high spin ferric heme”36 corresponds to a brown heme color.37 The 630 nm absorption peak also exists in both acid free heme monomer (fig. 2.5., part B) (heme = iron protoporphyrin IX) and in acid aqueous heme dimers38 (heme dimer = two heme molecules/monomers bound to each other). The 630 nm band is present in the bloodstain spectra and probably in the crystal spectrum as well, for otherwise it would not have been specified by Adler, Cameron, and another specialist, as old/denatured acid methemoglobin, as reported by Heller.39 In a drying solution, dissolved acid methemoglobin can form a (translucent) crystal, of which the crystal structure can be determined by X-ray crystallography.40 More denotations of blood material yielding these various spectra are in Heller and Adler’s Applied Optics article: “Thermodynamically the latter fibrils would be expected to show the spectrum of a fully oxidized denatured met-hemoglobin, i.e., a so-called perturbed acid met-hemoglobin7” 41, and “In our opinion the spectral data taken in aggregate are positive in confirming the presence of perturbed acid met-hemoglobin species on the Shroud.”42 Here, the meaning of “perturbed” is not specified, but it seems to have the notion of ‘denatured’ (applying to a deformed/unfolded protein or 32 Used from R. Gilbert Jr. and M.M. Gilbert, Ultraviolet-visible reflectance and fluorescence spectra of the Shroud of Turin, Applied Optics, 19 (12) 1980, pp. 1930-1936, http://dx.doi.org/10.1364/AO.19.001930, fig. 14, with permission from the Optical Society of America (fair use). (The corresponding absorbance figure (Gilbert and Gilbert, 1989, fig. 15) is online on the OSA website at http://imagebank.osa.org/getImage.xqy?img=QC5sYXJnZSxhby0xOS0xMi0xOTMwLWcwMTU ) 33 “Acid methemoglobin has a characteristic absorption peak at 630-631 nm” (A Mansouri, AA Lurie, Methemoglobinemia, American journal of hematology, 1993, http://onlinelibrary.wiley.com/doi/10.1002/ajh.2830420104/abstract , 5th find at http://scholar.google.nl/scholar?hl=nl&q=absorption+acid+methemoglobin&lr 34 Austin & Drabkin, 1935, http://www.jbc.org/content/112/1/67.short p. 70 (p. 4 of pdf file) 35 Wintrobe’s Clinical Hematology, Vol. 1, 12th edition, 2009, p. 147; “characterized … by the extinction coefficients of the bands at 406, 500 and 630 mµ. […] The extinction coefficients were E 406 mM = 141 to 150; E 500mM = 9.2; and E 630 mM = 3.7, to be compared with the values of 155, 9.5, and 4.1 (13) reported for ammonium sulfate-fractioned human Hb+.” Steinhardt and Hiremath, A Comparison of the Resistance of Human and Horse Ferrihemoglobin to Acid Denaturation, The Journal of Biological Chemistry , Vol. 242, no 6, 1967, http://www.jbc.org/content/242/6/1294.full.pdf ; See for instance figure 48-2 (A = methemoglobin A) of http://www.drugswell.com/winow/+%20b19/+%20williams%20hematology- 2007/V.%20The%20Erythrocyte/48.%20Methemoglobinemia%20and%20Other%20Causes%20of%20Cyanosis.htm ; or see methemoglobin at pH 7 at http://www.pnas.org/content/97/7/2984/F1.expansion.html 36 Appleby et al., Leghemoglobin,1976, http://www.jbc.org/content/251/19/6090.full.pdf (soybean leghemoglobin is a heme-containing protein comparable to human methemoglobin) 37 cf. Adler, The origin and nature…, 1986, TOM 60-61; also one of the authors of “Crystal structure and ligand binding properties of the truncated hemoglobin from Geobacillus stearothermophilus”, 2007, http://www.sciencedirect.com/science/article/pii/S0003986106003560, wrote in a personal communication, July 25, 2013, that a brown color is typical of high spin hexacoordinate heme; “acid haematin” is called brown in Meldrum, 1931, http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1260779/pdf/biochemj01118-0060.pdf 38 T.J. Egan (co-author of Villiers et al, Speciation and structure…, 2007), personal communication, 25-7-2013 39 “Adler got on the phone right away and read the coordinates to his chosen specialist. The answer – old acid methemoglobin. Then we called Bruce Cameron … “… It’s old acid methemoglobin. …”” Heller, Report on…, 1983, p. 147 40 “Although met- and oxyhemoglobin were reported to have similar conformations in the crystalline state (10), the present result clearly shows that the conformations of these two hemoglobins in dissolved state are different […] x-ray crystallography which showed that the structure of acid methemoglobin is similar to that of oxyhemoglobin (10). 10= Perutz and Matthews, 1966, J. Mol. Biol. 21, 199-202” Heme-Spin Label Studies of Hemoglobin II. SPIN-LABELED OXY-AND DEOXYHEMOGLOBINS, Asakura andTamura, 1974 http://www.jbc.org/content/249/14/4504.full.pdf 41 Note 7 = B.F. Cameron and P. George, Biochimica et Biophysica Acta, 194 (1969): 16, now online at http://www.sciencedirect.com/science/article/pii/0005279569901743 42 Heller and Adler, Blood on the Shroud…, 1980, TOM p. 30-31 9 even a dissociated/lost protein and a remaining heme) and it is reminiscent of ‘turbidity’ (applying to the aggregation state of the heme compounds in solution). Fig. 2.4. Absorbance of methemoglobin at pH 1 to 12 (mirrored image) ©the American Society for Biochemistry and Molecular Biology43 2.1.1.2. Heme dimerization The polymerisation/aggregation state of heme determines the presence or (nearly) absence of the Soret band. Both a heme bound to its globin protein and a single free heme shows the Soret band (at 405 nm if the heme is H O-ligated44) although a 370 nm ‘denatured’ band appears when the globin 2 dissociates from its acid methemoglobin: the ratio of the height of the Soret peak to the Q and charge transfer peaks stays the same in these acid species; only when two hemes form a dimer, the Soret peak disappears,45 relative to the other bands: the ratio of the band intensities changes (fig. 2.5).46 43 This research was originally published in Journal of Biological Chemistry. J.H. Austin and D.L. Drabkin. Spectrophotometric Studies: III. Methemoglobin. Journal of Biological Chemistry, 1935; 112:67-88. © the American Society for Biochemistry and Molecular Biology. http://www.jbc.org/content/112/1/67.short p. 70: fig. 1 (p. 4 of pdf file); According to Cameron and George (1969) an absorbance spectrum of methemoglobin at pH 7.0 seems not entirely reliable if there was “exposure even briefly to a pH below 6.0”, and therefore “The common practice of reporting a methemoglobin spectrum at pH 7.0 or 7.4 is to be discouraged.” Cameron and George, 1969, http://www.sciencedirect.com/science/article/pii/0005279569901743 44 methemoglobin: Wintrobe’s Clinical Hematology, Vol I, 12th edition, 2009, p. 147; heme monomer: Cf. “To our surprise, the heme complex exhibited a sharp Soret band at 405 nm in benzene;” here, there is a surfactant-heme complex made from a pH 7.0 aqueous buffer solution that would contain an equilibrium of monomer heme and weakly bound pi- pi dimers. (the “surfactant-heme complex” can not be “decomposed”); in the pH 7.0 aqueous solution the species yielding the 393 nm Soret peak is not the assumed mu-oxo dimer (assumed because of Brown et al., 1970) but probably the – in 1997 still unknown - aqueous heme pi-pi dimer in equilibrium with the heme monomer, which yields the 405 nm peak. (Kamiya et al., Peroxidase activity and stability of surfactant-heme complex in nonaqueous media, 1997, http://link.springer.com/article/10.1023/A:1018403518763). Cf. Villiers et al., Speciation and structure…, 2007, p. 102, describing the assumption on the µ-oxo dimer, also in relation to the article of Brown et al. 1970; Villiers et al. also used “80% aqueous methanol, where Fe(III)PPIX is monomeric” p. 108.; “Spectra of aqueous Fe(III)PPIX […] show that the Soret band (393 nm) decreases with increasing concentration as expected.” p. 107 (Fig. S3A, for pH 8.06, shows the isobestic point at 347 nm which later is also used for other pH values; however, the peak shifts to the blue with increasing concentration, so the 393 nm value is not the identical value for monomer and dimer, and probably is an intermediate value of the mix). 45 “The fast measurements at low pH make it possible to distinguish between several steps in the spectral changes: A-> B, a small exeedingly fast reaction, accompanied by a red-shift of the Soret band; B -> C, expulsion of the heme from the protein (blue shift of the Soret band); C -> D, the dimerization reaction of the free heme (disappearance of the Soret 10