f0b969021db049738ad6eeb6a0d0a07d pmc_33807076.pdf 70c6f4124a8d2b74e5282ba7b311d20d95565a4c pmc_33807076.pdf ddd17caf670f8aae5b33f48b3626ecaeaa3fa0abac1e761e46c035bbe5b65aa6 pmc_33807076.pdf Title: Conformational Heterogeneity and Cooperative Effects of Mammalian ALOX15 Subject: Arachidonic acid lipoxygenases (ALOXs) have been suggested to function as monomeric enzymes, but more recent data on rabbit ALOX15 indicated that there is a dynamic monomer-dimer equilibrium in aqueous solution. In the presence of an active site ligand (the ALOX15 inhibitor RS7) rabbit ALOX15 was crystalized as heterodimer and the X-ray coordinates of the two monomers within the dimer exhibit subtle structural differences. Using native polyacrylamide electrophoresis, we here observed that highly purified and predominantly monomeric rabbit ALOX15 and human ALOX15B are present in two conformers with distinct electrophoretic mobilities. In silico docking studies, molecular dynamics simulations, site directed mutagenesis experiments and kinetic measurements suggested that in aqueous solutions the two enzymes exhibit motional flexibility, which may impact the enzymatic properties. Keywords: lipoxygenases; crystal structure; protein–protein interactions; cooperative effects; molecular dynamics Author: Igor Ivanov, Alejandro Cruz, Alexander Zhuravlev, Almerinda Di Venere, Eleonora Nicolai, Sabine Stehling, José M. Lluch, Àngels González-Lafont and Hartmut Kuhn Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Thu Mar 25 09:57:35 2021 CET ModDate: Thu Mar 25 11:27:49 2021 CET Custom Metadata: no Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 18 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 33088636 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- SBMXUO+VnURWPalladioL Type 1 Custom yes yes no 10 0 QCRZYJ+URWPalladioL-Ital Type 1 Custom yes yes no 15 0 DGQQZW+URWPalladioL-Bold Type 1 Custom yes yes no 20 0 YGBAWR+URWPalladioL-Roma Type 1 Custom yes yes no 24 0 EPTQDJ+VnURWPalladioL-Bold Type 1 Custom yes yes no 28 0 IRWHZX+TimesNewRomanPSMT Type 1C WinAnsi yes yes no 36 0 CKUPDE+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 39 0 MCBCCI+SourceSansRoman-Regular Type 1C Custom yes yes no 61 0 MCBCCM+SourceSansRoman.613wght Type 1C Custom yes yes no 65 0 MCBCCO+SourceSansRoman-Bold Type 1C Custom yes yes no 69 0 VVSYYB+EURM10 Type 1 Builtin yes yes no 81 0 RYQVMQ+CMSY10 Type 1 Builtin yes yes no 99 0 XIRVNA+EURB10 Type 1 Builtin yes yes no 131 0 WWNSGS+URWPalladioL-BoldItal Type 1 Custom yes yes no 135 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2024-10-02 03:24:28 CEST RepresentationInformation: pmc_33807076.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2023-03-31 12:09:33 CEST Size: 33088636 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 431 FreeObjects: 9 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: section.1 Item: Title: Results Destination: section.2 Children: Item: Title: Structural Characterization of ALOX15 Isoenzymes by Native PAGE Destination: subsection.2.1 Item: Title: Consequences of N-terminal Truncation and Point Mutations of His585 on Conformational Heterogeneity of Rabbit ALOX15 Destination: subsection.2.2 Item: Title: Impact of Point Mutations of Amino Acids Localized at the Inter-Monomer Contact Site on the Reaction Kinetics of Rabbit ALOX15 Destination: subsection.2.3 Item: Title: MD Simulation of the Structural Alterations of Rabbit ALOX15 Induced by Trp181Glu Exchange Destination: subsection.2.4 Item: Title: AA and LA Binding Modes Destination: subsection.2.5 Item: Title: Analysis of Pre-Catalytic Structures Destination: subsection.2.6 Item: Title: Discussion Destination: section.3 Children: Item: Title: Conformational Heterogeneity of ALOX15 Destination: subsection.3.1 Item: Title: Cooperativity of Rabbit ALOX15 Monomers during Lipoxygenase Reaction Destination: subsection.3.2 Item: Title: Materials and Methods Destination: section.4 Children: Item: Title: Chemicals Destination: subsection.4.1 Item: Title: Expression of Rabbit ALOX15 Destination: subsection.4.2 Item: Title: Expression of Human ALOX15B Destination: subsection.4.3 Item: Title: Purification of Rabbit ALOX15 and Human ALOX15B Destination: subsection.4.4 Item: Title: Estimation of the Hydrodynamic Radii for Wild-Type and Mutant ALOX15 Destination: subsection.4.5 Item: Title: Native PAGE Electrophoresis Destination: subsection.4.6 Item: Title: CD Measurements Destination: subsection.4.7 Item: Title: Guanidine Denaturation Studies. Destination: subsection.4.8 Item: Title: Rabbit ALOX15 Activity Assay (Purified Enzyme) Destination: subsection.4.9 Item: Title: Molecular Docking Simulations Destination: subsection.4.10 Item: Title: Molecular Dynamics (MD) Simulations Destination: subsection.4.11 Item: Title: Conclusions Destination: section.5 Item: Title: References Destination: section.5 Info: Title: Conformational Heterogeneity and Cooperative Effects of Mammalian ALOX15 Author: Igor Ivanov, Alejandro Cruz, Alexander Zhuravlev, Almerinda Di Venere, Eleonora Nicolai, Sabine Stehling, José M. Lluch, Àngels González-Lafont and Hartmut Kuhn Subject: Arachidonic acid lipoxygenases (ALOXs) have been suggested to function as monomeric enzymes, but more recent data on rabbit ALOX15 indicated that there is a dynamic monomer-dimer equilibrium in aqueous solution. In the presence of an active site ligand (the ALOX15 inhibitor RS7) rabbit ALOX15 was crystalized as heterodimer and the X-ray coordinates of the two monomers within the dimer exhibit subtle structural differences. Using native polyacrylamide electrophoresis, we here observed that highly purified and predominantly monomeric rabbit ALOX15 and human ALOX15B are present in two conformers with distinct electrophoretic mobilities. In silico docking studies, molecular dynamics simulations, site directed mutagenesis experiments and kinetic measurements suggested that in aqueous solutions the two enzymes exhibit motional flexibility, which may impact the enzymatic properties. Keywords: lipoxygenases; crystal structure; protein protein interactions; cooperative effects; molecular dynamics Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Thu Mar 25 09:57:35 CET 2021 ModDate: Thu Mar 25 11:27:49 CET 2021 ID: 0x3e743ee08810f7e877f16eadd153efce, 0x3e743ee08810f7e877f16eadd153efce Filters: FilterPipeline: FlateDecode Fonts: Type1: Font: BaseFont: VVSYYB+EURM10 FontSubset: true FirstChar: 11 LastChar: 22 FontDescriptor: FontName: VVSYYB+EURM10 Flags: Symbolic FontBBox: -32, -243, 1060, 720 FontFile: true Font: BaseFont: RYQVMQ+CMSY10 FontSubset: true FirstChar: 0 LastChar: 36 FontDescriptor: FontName: RYQVMQ+CMSY10 Flags: Symbolic FontBBox: -29, -960, 1116, 775 FontFile: true Font: BaseFont: XIRVNA+EURB10 FontSubset: true FirstChar: 22 LastChar: 22 FontDescriptor: FontName: XIRVNA+EURB10 Flags: Symbolic FontBBox: -30, -247, 1178, 723 FontFile: true Font: BaseFont: DGQQZW+URWPalladioL-Bold FontSubset: true FirstChar: 3 LastChar: 197 FontDescriptor: FontName: DGQQZW+URWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: WWNSGS+URWPalladioL-BoldItal FontSubset: true FirstChar: 47 LastChar: 116 FontDescriptor: FontName: WWNSGS+URWPalladioL-BoldItal Flags: Symbolic FontBBox: -170, -300, 1073, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: YGBAWR+URWPalladioL-Roma FontSubset: true FirstChar: 2 LastChar: 246 FontDescriptor: FontName: YGBAWR+URWPalladioL-Roma Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: SBMXUO+VnURWPalladioL FontSubset: true FirstChar: 128 LastChar: 226 FontDescriptor: FontName: SBMXUO+VnURWPalladioL Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: EPTQDJ+VnURWPalladioL-Bold FontSubset: true FirstChar: 128 LastChar: 178 FontDescriptor: FontName: EPTQDJ+VnURWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: QCRZYJ+URWPalladioL-Ital FontSubset: true FirstChar: 2 LastChar: 122 FontDescriptor: FontName: QCRZYJ+URWPalladioL-Ital Flags: Symbolic FontBBox: -170, -305, 1010, 941 FontFile: true EncodingDictionary: Differences: true Pages: Page: Sequence: 1 Annotations: Annotation: Subtype: Link Rect: 35, 759, 181, 795 Annotation: Subtype: Link Rect: 515, 759, 560, 795 Annotation: Subtype: Link Rect: 94, 662, 105, 675 Annotation: Subtype: Link Rect: 186, 662, 197, 675 Annotation: Subtype: Link Rect: 500, 662, 511, 675 Annotation: Subtype: Link Rect: 195, 649, 206, 663 Annotation: Subtype: Link Rect: 333, 649, 344, 663 Annotation: Subtype: Link Rect: 445, 649, 456, 663 Annotation: Subtype: Link Rect: 55, 358, 154, 367 Annotation: Subtype: Link Rect: 35, 346, 77, 355 Annotation: Subtype: Link Rect: 35, 162, 97, 184 Annotation: Subtype: Link Rect: 153, 89, 154, 98 Annotation: Subtype: Link Rect: 35, 77, 147, 86 Annotation: Subtype: Link Rect: 332, 232, 339, 241 ActionDest: 172 Annotation: Subtype: Link Rect: 341, 232, 348, 241 ActionDest: 172 Annotation: Subtype: Link Rect: 301, 195, 308, 204 ActionDest: 172 Annotation: Subtype: Link Rect: 407, 170, 414, 179 ActionDest: 172 Annotation: Subtype: Link Rect: 415, 169, 422, 179 ActionDest: 172 Annotation: Subtype: Link Rect: 249, 94, 256, 103 ActionDest: 172 Annotation: Subtype: Link Rect: 259, 94, 266, 103 ActionDest: 172 Annotation: Subtype: Link Rect: 536, 82, 543, 91 ActionDest: 172 Annotation: Subtype: Link Rect: 544, 82, 556, 91 ActionDest: 172 Annotation: Subtype: Link Rect: 139, 17, 278, 28 Annotation: Subtype: Link Rect: 420, 17, 558, 28 Annotation: Subtype: Link Rect: 35, 458, 87, 477 Page: Sequence: 2 Annotations: Annotation: Subtype: Link Rect: 210, 674, 222, 683 ActionDest: 172 Annotation: Subtype: Link Rect: 223, 674, 235, 683 ActionDest: 172 Annotation: Subtype: Link Rect: 414, 661, 426, 670 ActionDest: 172 Annotation: Subtype: Link Rect: 252, 599, 264, 608 ActionDest: 172 Annotation: Subtype: Link Rect: 461, 511, 473, 520 ActionDest: 172 Annotation: Subtype: Link Rect: 495, 485, 507, 495 ActionDest: 172 Annotation: Subtype: Link Rect: 407, 460, 419, 469 ActionDest: 172 Annotation: Subtype: Link Rect: 348, 423, 360, 432 ActionDest: 172 Annotation: Subtype: Link Rect: 265, 398, 277, 407 ActionDest: 172 Annotation: Subtype: Link Rect: 278, 398, 290, 407 ActionDest: 172 Annotation: Subtype: Link Rect: 212, 372, 224, 382 ActionDest: 172 Annotation: Subtype: Link Rect: 279, 347, 291, 356 ActionDest: 172 Annotation: Subtype: Link Rect: 217, 322, 229, 331 ActionDest: 172 Annotation: Subtype: Link Rect: 232, 322, 244, 331 ActionDest: 172 Annotation: Subtype: Link Rect: 249, 169, 256, 181 ActionDest: 85 Annotation: Subtype: Link Rect: 241, 156, 248, 168 ActionDest: 85 Annotation: Subtype: Link Rect: 490, 134, 502, 143 ActionDest: 172 Annotation: Subtype: Link Rect: 221, 96, 233, 105 ActionDest: 172 Page: Sequence: 3 Annotations: Annotation: Subtype: Link Rect: 531, 247, 543, 256 ActionDest: 172 Annotation: Subtype: Link Rect: 544, 247, 556, 256 ActionDest: 172 Annotation: Subtype: Link Rect: 297, 157, 303, 169 ActionDest: 85 Annotation: Subtype: Link Rect: 490, 106, 497, 118 ActionDest: 85 Annotation: Subtype: Link Rect: 393, 81, 400, 93 ActionDest: 85 Page: Sequence: 4 Annotations: Annotation: Subtype: Link Rect: 200, 682, 207, 694 ActionDest: 85 Annotation: Subtype: Link Rect: 241, 621, 248, 633 ActionDest: 85 Annotation: Subtype: Link Rect: 293, 598, 304, 608 ActionDest: 172 Annotation: Subtype: Link Rect: 305, 598, 317, 608 ActionDest: 172 Annotation: Subtype: Link Rect: 486, 571, 493, 583 ActionDest: 103 Annotation: Subtype: Link Rect: 235, 483, 242, 495 ActionDest: 103 Annotation: Subtype: Link Rect: 216, 448, 228, 457 ActionDest: 172 Annotation: Subtype: Link Rect: 380, 445, 560, 457 Annotation: Subtype: Link Rect: 166, 433, 261, 445 Annotation: Subtype: Link Rect: 200, 345, 207, 357 ActionDest: 103 Annotation: Subtype: Link Rect: 363, 345, 370, 357 ActionDest: 103 Annotation: Subtype: Link Rect: 355, 332, 362, 344 ActionDest: 103 Annotation: Subtype: Link Rect: 321, 320, 328, 332 ActionDest: 103 Annotation: Subtype: Link Rect: 229, 207, 236, 219 ActionDest: 103 Annotation: Subtype: Link Rect: 210, 144, 217, 156 ActionDest: 103 Annotation: Subtype: Link Rect: 289, 144, 296, 156 ActionDest: 103 Annotation: Subtype: Link Rect: 340, 109, 352, 118 ActionDest: 172 Annotation: Subtype: Link Rect: 228, 84, 240, 93 ActionDest: 172 Annotation: Subtype: Link Rect: 340, 56, 347, 68 ActionDest: 103 Page: Sequence: 5 Page: Sequence: 6 Annotations: Annotation: Subtype: Link Rect: 445, 732, 452, 744 ActionDest: 103 Annotation: Subtype: Link Rect: 545, 594, 552, 606 ActionDest: 103 Annotation: Subtype: Link Rect: 258, 584, 270, 593 ActionDest: 172 Annotation: Subtype: Link Rect: 238, 556, 245, 568 ActionDest: 103 Annotation: Subtype: Link Rect: 320, 519, 327, 531 ActionDest: 103 Annotation: Subtype: Link Rect: 269, 481, 276, 493 ActionDest: 103 Annotation: Subtype: Link Rect: 231, 471, 243, 480 ActionDest: 175 Annotation: Subtype: Link Rect: 260, 459, 272, 468 ActionDest: 175 Annotation: Subtype: Link Rect: 542, 368, 549, 380 ActionDest: 103 Annotation: Subtype: Link Rect: 233, 272, 245, 281 ActionDest: 172 Annotation: Subtype: Link Rect: 243, 194, 250, 206 ActionDest: 117 Annotation: Subtype: Link Rect: 266, 159, 278, 168 ActionDest: 172 Page: Sequence: 7 Annotations: Annotation: Subtype: Link Rect: 273, 289, 280, 301 ActionDest: 128 Annotation: Subtype: Link Rect: 296, 228, 308, 238 ActionDest: 175 Annotation: Subtype: Link Rect: 450, 188, 457, 200 ActionDest: 128 Annotation: Subtype: Link Rect: 491, 188, 498, 200 ActionDest: 117 Annotation: Subtype: Link Rect: 294, 141, 306, 150 ActionDest: 175 Page: Sequence: 8 Annotations: Annotation: Subtype: Link Rect: 277, 463, 284, 475 ActionDest: 139 Annotation: Subtype: Link Rect: 200, 387, 207, 400 ActionDest: 139 Annotation: Subtype: Link Rect: 258, 362, 265, 374 ActionDest: 139 Page: Sequence: 9 Page: Sequence: 10 Annotations: Annotation: Subtype: Link Rect: 534, 720, 541, 732 ActionDest: 139 Annotation: Subtype: Link Rect: 267, 682, 274, 694 ActionDest: 139 Annotation: Subtype: Link Rect: 475, 672, 487, 681 ActionDest: 172 Annotation: Subtype: Link Rect: 286, 622, 298, 631 ActionDest: 175 Annotation: Subtype: Link Rect: 331, 594, 338, 608 ActionDest: 139 Annotation: Subtype: Link Rect: 325, 556, 332, 568 ActionDest: 139 Annotation: Subtype: Link Rect: 326, 521, 338, 530 ActionDest: 175 Annotation: Subtype: Link Rect: 434, 496, 446, 505 ActionDest: 175 Annotation: Subtype: Link Rect: 323, 484, 335, 493 ActionDest: 175 Annotation: Subtype: Link Rect: 229, 443, 236, 455 ActionDest: 139 Annotation: Subtype: Link Rect: 203, 408, 215, 417 ActionDest: 172 Annotation: Subtype: Link Rect: 449, 380, 456, 392 ActionDest: 139 Annotation: Subtype: Link Rect: 289, 354, 296, 367 ActionDest: 153 Annotation: Subtype: Link Rect: 408, 320, 420, 329 ActionDest: 175 Annotation: Subtype: Link Rect: 420, 320, 432, 329 ActionDest: 175 Annotation: Subtype: Link Rect: 219, 180, 226, 192 ActionDest: 139 Annotation: Subtype: Link Rect: 201, 104, 208, 116 ActionDest: 139 Annotation: Subtype: Link Rect: 192, 90, 199, 104 ActionDest: 153 Page: Sequence: 11 Annotations: Annotation: Subtype: Link Rect: 234, 360, 241, 372 ActionDest: 153 Annotation: Subtype: Link Rect: 487, 347, 494, 359 ActionDest: 128 Annotation: Subtype: Link Rect: 521, 334, 528, 346 ActionDest: 153 Annotation: Subtype: Link Rect: 317, 309, 324, 321 ActionDest: 128 Annotation: Subtype: Link Rect: 297, 248, 309, 257 ActionDest: 175 Annotation: Subtype: Link Rect: 310, 248, 322, 257 ActionDest: 175 Annotation: Subtype: Link Rect: 206, 210, 218, 219 ActionDest: 172 Annotation: Subtype: Link Rect: 309, 210, 321, 219 ActionDest: 172 Annotation: Subtype: Link Rect: 358, 198, 370, 207 ActionDest: 175 Page: Sequence: 12 Annotations: Annotation: Subtype: Link Rect: 542, 707, 549, 719 ActionDest: 117 Annotation: Subtype: Link Rect: 238, 647, 250, 656 ActionDest: 172 Annotation: Subtype: Link Rect: 251, 647, 263, 656 ActionDest: 172 Annotation: Subtype: Link Rect: 257, 607, 264, 619 ActionDest: 85 Annotation: Subtype: Link Rect: 200, 581, 207, 594 ActionDest: 103 Annotation: Subtype: Link Rect: 345, 559, 357, 568 ActionDest: 172 Annotation: Subtype: Link Rect: 503, 534, 515, 543 ActionDest: 172 Annotation: Subtype: Link Rect: 319, 456, 326, 468 ActionDest: 103 Annotation: Subtype: Link Rect: 482, 320, 494, 329 ActionDest: 172 Annotation: Subtype: Link Rect: 232, 280, 239, 292 ActionDest: 117 Annotation: Subtype: Link Rect: 302, 270, 314, 279 ActionDest: 172 Annotation: Subtype: Link Rect: 235, 230, 242, 242 ActionDest: 128 Annotation: Subtype: Link Rect: 275, 230, 282, 242 ActionDest: 117 Annotation: Subtype: Link Rect: 364, 217, 371, 229 ActionDest: 128 Annotation: Subtype: Link Rect: 396, 167, 403, 179 ActionDest: 103 Page: Sequence: 13 Annotations: Annotation: Subtype: Link Rect: 374, 735, 386, 744 ActionDest: 175 Annotation: Subtype: Link Rect: 387, 735, 399, 744 ActionDest: 175 Annotation: Subtype: Link Rect: 531, 634, 543, 643 ActionDest: 172 Annotation: Subtype: Link Rect: 543, 634, 555, 643 ActionDest: 175 Annotation: Subtype: Link Rect: 189, 534, 201, 543 ActionDest: 172 Annotation: Subtype: Link Rect: 492, 206, 504, 215 ActionDest: 172 Page: Sequence: 14 Annotations: Annotation: Subtype: Link Rect: 477, 367, 484, 381 ActionDest: 103 Page: Sequence: 15 Annotations: Annotation: Subtype: Link Rect: 510, 719, 522, 728 ActionDest: 175 Annotation: Subtype: Link Rect: 493, 493, 505, 502 ActionDest: 175 Annotation: Subtype: Link Rect: 402, 480, 414, 489 ActionDest: 175 Annotation: Subtype: Link Rect: 415, 480, 427, 489 ActionDest: 175 Annotation: Subtype: Link Rect: 270, 266, 282, 275 ActionDest: 175 Annotation: Subtype: Link Rect: 342, 266, 354, 275 ActionDest: 175 Annotation: Subtype: Link Rect: 357, 254, 369, 263 ActionDest: 172 Annotation: Subtype: Link Rect: 375, 241, 387, 250 ActionDest: 175 Annotation: Subtype: Link Rect: 430, 241, 442, 250 ActionDest: 175 Annotation: Subtype: Link Rect: 220, 216, 232, 225 ActionDest: 175 Annotation: Subtype: Link Rect: 233, 216, 245, 225 ActionDest: 175 Annotation: Subtype: Link Rect: 402, 179, 414, 188 ActionDest: 175 Annotation: Subtype: Link Rect: 350, 128, 362, 137 ActionDest: 175 Annotation: Subtype: Link Rect: 362, 128, 374, 137 ActionDest: 175 Page: Sequence: 16 Annotations: Annotation: Subtype: Link Rect: 364, 659, 376, 669 ActionDest: 175 Annotation: Subtype: Link Rect: 258, 647, 270, 656 ActionDest: 175 Annotation: Subtype: Link Rect: 255, 597, 267, 606 ActionDest: 175 Annotation: Subtype: Link Rect: 259, 509, 271, 518 ActionDest: 172 Annotation: Subtype: Link Rect: 524, 496, 536, 505 ActionDest: 175 Annotation: Subtype: Link Rect: 248, 484, 260, 493 ActionDest: 175 Page: Sequence: 17 Annotations: Annotation: Subtype: Link Rect: 95, 707, 133, 717 Annotation: Subtype: Link Rect: 59, 684, 97, 694 Annotation: Subtype: Link Rect: 145, 661, 182, 671 Annotation: Subtype: Link Rect: 313, 637, 351, 648 Annotation: Subtype: Link Rect: 59, 615, 97, 625 Annotation: Subtype: Link Rect: 103, 615, 139, 625 Annotation: Subtype: Link Rect: 404, 591, 441, 602 Annotation: Subtype: Link Rect: 140, 558, 178, 568 Annotation: Subtype: Link Rect: 184, 558, 220, 568 Annotation: Subtype: Link Rect: 350, 534, 388, 545 Annotation: Subtype: Link Rect: 394, 534, 430, 545 Annotation: Subtype: Link Rect: 159, 500, 197, 510 Annotation: Subtype: Link Rect: 203, 500, 239, 510 Annotation: Subtype: Link Rect: 324, 465, 361, 476 Annotation: Subtype: Link Rect: 367, 465, 403, 476 Annotation: Subtype: Link Rect: 104, 431, 142, 441 Annotation: Subtype: Link Rect: 224, 407, 261, 418 Annotation: Subtype: Link Rect: 267, 407, 304, 418 Annotation: Subtype: Link Rect: 205, 373, 243, 384 Annotation: Subtype: Link Rect: 261, 350, 299, 361 Annotation: Subtype: Link Rect: 113, 316, 151, 326 Annotation: Subtype: Link Rect: 157, 316, 193, 326 Annotation: Subtype: Link Rect: 350, 292, 388, 303 Annotation: Subtype: Link Rect: 394, 292, 430, 303 Annotation: Subtype: Link Rect: 270, 258, 307, 269 Annotation: Subtype: Link Rect: 519, 235, 557, 246 Annotation: Subtype: Link Rect: 59, 224, 95, 234 Annotation: Subtype: Link Rect: 113, 189, 151, 200 Annotation: Subtype: Link Rect: 157, 189, 193, 200 Annotation: Subtype: Link Rect: 306, 154, 343, 165 Annotation: Subtype: Link Rect: 323, 131, 360, 142 Annotation: Subtype: Link Rect: 366, 131, 402, 142 Annotation: Subtype: Link Rect: 228, 109, 265, 119 Annotation: Subtype: Link Rect: 271, 109, 307, 119 Annotation: Subtype: Link Rect: 136, 74, 173, 85 Page: Sequence: 18 Annotations: Annotation: Subtype: Link Rect: 95, 722, 133, 732 Annotation: Subtype: Link Rect: 504, 698, 541, 709 Annotation: Subtype: Link Rect: 417, 675, 454, 686 Annotation: Subtype: Link Rect: 461, 675, 497, 686 Annotation: Subtype: Link Rect: 431, 652, 468, 663 Annotation: Subtype: Link Rect: 520, 640, 557, 651 Annotation: Subtype: Link Rect: 59, 630, 95, 640 Annotation: Subtype: Link Rect: 222, 594, 260, 605 Annotation: Subtype: Link Rect: 413, 560, 451, 571 Annotation: Subtype: Link Rect: 127, 526, 164, 536 Annotation: Subtype: Link Rect: 238, 492, 275, 502 Annotation: Subtype: Link Rect: 436, 468, 473, 479 Annotation: Subtype: Link Rect: 480, 468, 516, 479 Annotation: Subtype: Link Rect: 483, 445, 520, 456 Annotation: Subtype: Link Rect: 175, 423, 213, 433 Annotation: Subtype: Link Rect: 423, 399, 460, 410 Annotation: Subtype: Link Rect: 344, 376, 382, 387 Annotation: Subtype: Link Rect: 437, 330, 474, 341 Annotation: Subtype: Link Rect: 481, 330, 517, 341 Annotation: Subtype: Link Rect: 171, 308, 209, 318 Annotation: Subtype: Link Rect: 215, 308, 251, 318 Annotation: Subtype: Link Rect: 140, 273, 178, 283 Annotation: Subtype: Link Rect: 166, 250, 203, 260 Annotation: Subtype: Link Rect: 210, 250, 246, 260 Annotation: Subtype: Link Rect: 290, 226, 327, 237 Annotation: Subtype: Link Rect: 324, 203, 362, 214 Annotation: Subtype: Link Rect: 467, 180, 504, 191 Annotation: Subtype: Link Rect: 510, 180, 546, 191 Annotation: Subtype: Link Rect: 220, 146, 257, 157 Annotation: Subtype: Link Rect: 332, 123, 370, 134 Annotation: Subtype: Link Rect: 470, 111, 508, 122 Annotation: Subtype: Link Rect: 390, 88, 427, 99 Checksum: d432f1e2 Type: CRC32 Checksum: f0b969021db049738ad6eeb6a0d0a07d Type: MD5 Checksum: 70c6f4124a8d2b74e5282ba7b311d20d95565a4c Type: SHA-1 Checksum: ddd17caf670f8aae5b33f48b3626ecaeaa3fa0abac1e761e46c035bbe5b65aa6 Type: SHA-256