Analytical approaches to the study of hyaluronan and hyaluronidases: Development and application of hyphenated chromatographic and electrophoretic methods
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| Format: | Thesis Book |
| Language: | English |
| Published: |
2013
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| Subjects: | |
| Online Access: | Volltext https://nbn-resolving.org/urn:nbn:de:bvb:355-epub-279496 Inhaltsverzeichnis |
| Physical Description: | XVI, 213 S. Ill., graph. Darst. |
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| 245 | 1 | 0 | |a Analytical approaches to the study of hyaluronan and hyaluronidases |b Development and application of hyphenated chromatographic and electrophoretic methods |c von Martin Rothenhöfer |
| 264 | 1 | |c 2013 | |
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| adam_text | I
CONTENTS
CONTENTS I
CURRICULUM VITAE VII
PUBLICATIONS, ORAL
PRESENTATIONS, AND POSTERS IX
ACKNOWLEDGEMENTS AND
DECLARATION OF COLLABORATIONS XI
ABBREVIATIONS ! XIII
1 GENERAL INTRODUCTION 1
1.1 HYALURONAN 1
1.1.1 STRUCTURE AND PROPERTIES: A CHEMICAL POINT OF VIEW
1.1.2 OCCURRENCE AND PHYSIOLOGICAL
RELEVANCE: A BIOLOGICAL
POINT OF VIEW
1.2 HYALURONIDASES AND HYALURONAN
METABOLISM
1.2.1 OCCURRENCE AND CLASSIFICATION
OF HYALURONIDASES
1.2.2 HUMAN HYALURONIDASES 6
1.2.3 HYALURONAN METABOLISM 8
1.2.4 HYALURONAN RECEPTORS 9
1.2.5 EFFECTS OF HYALURONAN
AND ITS
OLIGOSACCHARIDE FRAGMENTS: A MATTER OF SIZE? 10
1.3 ANALYTICAL APPROACHES TO THE STUDY OF HYALURONAN, HYALURONAN
OLIGOSACCHARIDES,
AND HYALURONIDASES 11
1.3.1 CONVENTIONAL METHODS 11
1.3.2 CHROMATOGRAPHIC METHODS 13
1.3.3 ELECTROPHORETIC METHODS 14
1.3.4 OTHER METHODS 15
1.4 REFERENCES 15
2 SCOPE AND OBJECTIVES 27
3 CZE-ESI-TOF-MS FOR THE
FAST ANALYSIS OF SMALL
HYALURONAN OLIGOSACCHARIDES 29
3.1 INTRODUCTION 29
3.2 MATERIALS AND METHODS 30
3.2.1 OLIGOSACCHARIDE STANDARDS AND
SAMPLE PREPARATION 30
3.2.2 EOF MARKERS AND
INTERNAL STANDARD 30
3.2.3 PREPARATION OF THE BACKGROUND ELECTROLYTES 32
3.2.4 INSTRUMENTATION, CAPILLARY MATERIAL, AND SEPARATION CONDITIONS FOR
CZE-UV
EXPERIMENTS 32
3.2.5 INSTRUMENTATION AND CAPILLARY MATERIAL FOR CZE-ESI-TOF-MS 32
3.2.6 SHEATH LIQUID,
MS PARAMETER SETTINGS, AND MASS
CALIBRATION 33
HTTP://D-NB.INFO/1049959248
II CONTENTS
3.2.7 MANUAL SAMPLE INJECTION 36
3.3 RESULTS AND DISCUSSION 36
3.3.1 PRELIMINARY TESTS USING
CZE-UV 36
3.3.2 OPTIMIZATION OF THE INJECTION RONE LENGTH 38
3.3.3 CAPILLARY LENGTH AND
ELECTRIC FIELD STRENGTH 39
3.3.4 CAPILLARY INNER DIAMETER 41
3.3.5 CONCENTRATION AND
PH OF
THE BACKGROUND ELECTROLYTE 43
3.3.6 CHARACTERIZATION OF THE OPTIMIZED METHOD . 45
3.3.7 APPLICATION TO A COMPLEX HYALURONAN OLIGOSACCHARIDE
MIXTURE 47
3.3.8 ACARBOSE AS INTERNAL
STANDARD FOR AT-LINE ANALYSIS 48
3.4 SUMMARY AND
CONCLUSION 51
3.5 REFERENCES 52
4 HPAEC-PAD FOR SENSITIVE DETERMINATION OF HYALURONAN OLIGOSACCHARIDES
UP TO
10 KDA 55
4.1 INTRODUCTION 55
4.2 MATERIALS AND METHODS 56
4.2.1 OLIGOSACCHARIDE STANDARDS AND SAMPLE
PREPARATION 56
4.2.2 PREPARATION AND
STORAGE OF ELUENTS 56
4.2.3 INSTRUMENTATION AND CHROMATOGRAPHY CONDITIONS 57
4.2.4 ELECTROCHEMICAL DETECTOR SETTINGS 57
4.2.5 ASSESSMENT OF ANALYTE STABILITY
BY CZE-ESI-TOF-MS 57
4.2.6 SIZE-DEPENDENT PRECIPITATION OF HYALURONAN FRAGMENTS 57
4.3 RESULTS AND DISCUSSION 58
4.3.1 OPTIMIZATION OF THE SODIUM HYDROXIDE CONCENTRATION 58
4.3.2 SODIUM ACETATE GRADIENT 59
4.3.3 COLUMN TEMPERATURE 61
4.3.4 COMPARISON OF THE CARBOPAC* PA100 AND THE CARBOPAC* PA200
COLUMN AT
DIFFERENT FLOW AND TEMPERATURE CONDITIONS 62
4.3.5 CHARACTERIZATION OF THE OPTIMIZED METHOD 63
4.3.6 ANALYSIS OF A COMPLEX MIXTURE OF HYALURONAN OLIGOSACCHARIDES 65
4.3.7 FORMATION OF ODD-NUMBERED OLIGOSACCHARIDES UNDER STRONGLY ALKALINE
CONDITIONS. 66
4.3.8 STUDIES ON THE SIZE-DEPENDENT PRECIPITATION OF HYALURONAN
FRAGMENTS IN THE
TURBIDIMETRIC HYALURONIDASE ACTIVITY ASSAY 69
4.4 SUMMARY AND CONCLUSION 70
4.5 REFERENCES 71
5 PREPARATION OF PURIFIED HYALURONAN OLIGOSACCHARIDES AND PLANAR
CHROMATOGRAPHY FOR QUALITY CONTROL 75
5.1 INTRODUCTION 75
5.2 MATERIALS AND METHODS 76
5.2.1 STANDARD ANALYTES
AND PREPARATION OF OLIGOSACCHARIDES 76
CONTENTS TIL
5.2.2 HPAEC-PAD OF HA
OLIGOSACCHARIDE MIXTURES FROM ENZYMATIC DIGESTION 77
5.2.3 COLORIMETRIC ESTIMATION OF OLIGOSACCHARIDE CONCENTRATION 77
5.2.4 STATIONARY AND
MOBILE PHASES 77
5.2.5 SAMPLE APPLICATION
TECHNIQUE AND CHROMATOGRAPHY CONDITIONS 78
5.2.6 POST-CHROMATOGRAPHIC DERIVATIZATION 78
5.2.7 DOCUMENTATION, MEASUREMENT OF ABSORBANCE SPECTRA, DENSITOMETRIC
QUANTIFICATION, AND DATA
PROCESSING 79
5.2.8 COUPLING TO MASS SPECTROMETRY 79
5.3 RESULTS AND
DISCUSSION 81
5.3.1 OPTIMIZATION OF OLIGOSACCHARIDE PREPARATION BY ENZYMATIC DIGESTION
81
5.3.2 CHOICE AND OPTIMIZATION OF MOBILE PHASE FOR HPTLC 82
5.3.3 COMPARISON OF STATIONARY PHASE MATERIALS AND POST-CHROMATOGRAPHIC
DERIVATIZATION METHODS 82
5.3.4 STABILITY OF THE ANALYTES 85
5.3.5 OPTIMIZATION OF THE QUANTITATIVE DETECTION METHOD 85
5.3.6 CHARACTERIZATION OF THE OPTIMIZED QUANTITATIVE METHOD 87
5.3.7 ROBUSTNESS 90
5.3.8 THIN LAYER CHROMATOGRAPHY COUPLED TO MASS SPECTROMETRY 92
5.4 SUMMARY AND CONCLUSION 94
5.5 REFERENCES 95
6 PRODUCT SPECTRA OF HYALURONIDASES FROM BOVINE TESTES AND FROM
STREPTOCOCCUS
AGALACTIAE 97
6.1 INTRODUCTION 97
6.2 MATERIALS AND METHODS 98
6.2.1 ENZYMES AND SUBSTRATES 98
6.2.2 INCUBATION MIXTURES 99
6.2.3 HPAEC-PAD 99
6.2.4 FAST AT-LINE CZE-ESI-TOF-MS 100
6.2.5 SATURATION EXPERIMENT 100
6.3 RESULTS AND
DISCUSSION 101
6.3.1 DEGRADATION OF A LOW MOLECULAR WEIGHT HYALURONAN OLIGOSACCHARIDE
MIXTURE BY
BOVINE TESTICULAR HYALURONIDASE 101
6.3.2 SIZE-DEPENDENT DEGRADATION OF HYALURONAN BY BOVINE TESTICULAR
HYALURONIDASE.... 103
6.3.3 FAST AT-LINE ANALYSIS OF HYALURONAN OLIGOSACCHARIDE DEGRADATION BY
BOVINE
TESTICULAR HYALURONIDASE 104
6.3.4 DEGRADATION OF A LOW MOLECULAR WEIGHT HYALURONAN OLIGOSACCHARIDE
MIXTURE BY
HYALURONIDASE FROM STREPTOCOCCUS AGALACTIAE 108
6.3.5 SIZE-DEPENDENT DEGRADATION OF HYALURONAN BY HYALURONIDASE FROM
STREPTOCOCCUS AGALACTIAE 110
6.3.6 FAST AT-LINE ANALYSIS OF HYALURONAN OLIGOSACCHARIDE DEGRADATION BY
HYALURONIDASE FROM STREPTOCOCCUS AGALACTIAE ILL
IV
CONTENTS
6.3.7 SATURATION EXPERIMENT USING THE MINIMAL SUBSTRATE (N
2
) FOR STREPTOCOCCUS
AGALACTIAE HYALURONIDASE 116
6.4 SUMMARY AND CONCLUSION 118
6.5 REFERENCES 118
7 IN VITRO STUDIES
ON RECOMBINANT HUMAN PH-20, PEG-PH-20, AND
HYAL-1 121
7.1 INTRODUCTION 121
7.2 MATERIALS AND METHODS 122
7.2.1 ENZYMES, ACTIVITY
UNITS, SUBSTRATES, AND PUTATIVE INHIBITORS 122
7.2.2 DETERMINATION OF PROTEIN CONTENT 122
7.2.3 INCUBATION MIXTURES 123
7.2.4 COLORIMETRIC HYALURONIDASE ACTIVITY ASSAY 123
7.2.5 TURBIDIMETRIC HYALURONIDASE ACTIVITY ASSAY 123
7.2.6 HPAEC-PAD AND FAST AT-LINE
CZE-ESI-TOF-MS 124
7.3 RESULTS AND DISCUSSION 124
7.3.1 PROTEIN CONTENT AND ACTIVITY 124
7.3.2 INFLUENCE OF PH
ON ENZYMATIC
ACTIVITY 125
7.3.3 DEGRADATION OF A LOW MOLECULAR WEIGHT HYALURONAN OLIGOSACCHARIDE
MIXTURE BY
RHPH-20, PEG-RHPH-20,
AND BTH 127
7.3.4 FAST AT-LINE ANALYSIS OF HYALURONAN OLIGOSACCHARIDE DEGRADATION BY
RECOMBINANT
HUMAN PH-20 135
7.3.5 DEGRADATION OF A LOW MOLECULAR WEIGHT HYALURONAN OLIGOSACCHARIDE
MIXTURE BY
RECOMBINANT HUMAN HYAL-1 139
7.3.6 FAST AT-LINE ANALYSIS OF HYALURONAN OLIGOSACCHARIDE DEGRADATION BY
RECOMBINANT
HUMAN HYAL-1 141
7.3.7 THE QUEST FOR
INHIBITORS 146
7.4 SUMMARY AND
CONCLUSION 149
7.5 REFERENCES 149
8 ANALYSIS OF SYNOVIAL FLUID SAMPLES 153
8.1 INTRODUCTION 153
8.2 MATERIALS AND METHODS 153
8.2.1 ORIGIN OF SAMPLES 153
8.2.2 MACROSCOPIC CHARACTERIZATION AND
MICROSCOPY OF SMEARS 154
8.2.3 RHEOLOGY 154
8.2.4 DETERMINATION OF PROTEIN CONTENT 155
8.2.5 PREPARATION OF SAMPLES
FOR HYALURONAN ANALYSIS 155
8.2.6 ENZYMATIC DIGESTION OF HYALURONAN 155
8.2.7 COLORIMETRIC QUANTIFICATION 155
8.2.8 DETERMINATION OF HYALURONAN CONTENT BY CZE-ESI-TOF-MS 156
8.2.9 HPAEC-PAD OF DIGESTED
AND UNDIGESTED
SAMPLES 156
8.2.10 HYALURONAN ANALYSIS BY GEL ELECTROPHORESIS 156
CONTENTS V
8.2.11 ZYMOGRAPHY 158
8.3 RESULTS AND
DISCUSSION 159
8.3.1 MACROSCOPIC CHARACTERIZATION AND MICROSCOPIC FINDINGS 159
8.3.2 VISCOELASTIC PROPERTIES 161
8.3.3 PROTEIN CONTENT 165
8.3.4 TOTAL HYALURONAN CONTENT 165
8.3.5 SIZE DISTRIBUTION OF HYALURONAN 166
8.3.6 HYALURONIDASE ACTIVITY 170
8.4 SUMMARY AND CONCLUSION 171
8.5 REFERENCES 172
9 IN VITRO STUDIES ON THE INTERACTION OF HYALURONAN AND HYALURONAN
OLIGOSACCHARIDES WITH
HUMAN CELLS 175
9.1 INTRODUCTION 175
9.2 MATERIALS AND METHODS 176
9.2.1 CELL LINES
AND CELL
CULTURE CONDITIONS 176
9.2.2 TEST SUBSTANCES 177
9.2.3 FLOW CYTOMETRY 177
9.2.4 ELECTRIC CELL-SUBSTRATE IMPEDANCE SENSING (ECIS) OF ENDOTHELIAL
CELLS 178
9.2.5 CRYSTAL VIOLET CELL
PROLIFERATION ASSAY 179
9.2.6 COLLECTION AND ANALYSIS
OF WOUND MATERIAL 180
9.2.7 ANALYSIS OF CONDITIONED
CELL CULTURE MEDIA 181
9.3 RESULTS AND
DISCUSSION 181
9.3.1 FLOW CYTOMETRY 181
9.3.2 ELECTRIC WOUND HEALING
MODEL 183
9.3.3 PROLIFERATION OF HMEC-1
CELLS 184
9.3.4 ANALYSIS OF WOUND
MATERIAL 191
9.3.5 HPAEC-PAD
OF HYALURONAN OLIGOSACCHARIDE-SUPPLEMENTED CONDITIONED CELL
CULTURE MEDIA 192
9.4 SUMMARY AND CONCLUSION 193
9.5 REFERENCES 194
10 STUDIES ON POTENTIAL EFFECTS OF HYALURONAN AND ITS OLIGOSACCHARIDES
ON
ANGIOGENESIS IN THE CAM ASSAY 197
10.1 INTRODUCTION 197
10.2 MATERIALS AND METHODS 198
10.2.1 TEST SUBSTANCES 198
10.2.2 EGGS AND INCUBATION
CONDITIONS 198
10.2.3 OPENING OF THE CHICKEN
EGGS 198
10.2.4 APPLICATION OF THE TEST COMPOUNDS 200
10.2.5 STEREO MICROSCOPY
AND EVALUATION SCORE 201
10.2.6 PREPARATION, STAINING,
AND MICROSCOPY OF CHORIOALLANTOIC MEMBRANES 202
VI CONTENTS
10.3 RESULTS AND
DISCUSSION 202
10.3.1 SUITABILITY OF AGAROSE
RINGS 202
10.3.2 NORMAL CHORIOALLANTOIC MEMBRANES 202
10.3.3 IRRITATION OF THE CHORIOALLANTOIC MEMBRANE 203
10.3.4 SCORING OF EFFECTS 203
10.3.5 IMPACT OF HYALURONAN AND HYALURONAN
OLIGOSACCHARIDES ON ANGIOGENESIS 204
10.4 SUMMARY AND CONCLUSION 207
10.5 REFERENCES 207
11 SUMMARY AND OUTLOOK 209
11.1 METHOD DEVELOPMENT 209
11.2 IN VITRO STUDIES
ON HYALURONIDASES 211
11.3 BIOANALYTICAL AND
BIOLOGICAL INVESTIGATIONS 212
|
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| discipline | Chemie / Pharmazie |
| format | Thesis Book |
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| illustrated | Illustrated |
| indexdate | 2024-07-10T01:04:02Z |
| institution | BVB |
| language | English |
| oai_aleph_id | oai:aleph.bib-bvb.de:BVB01-027179986 |
| oclc_num | 876872289 |
| open_access_boolean | 1 |
| owner | DE-384 DE-473 DE-BY-UBG DE-703 DE-1051 DE-824 DE-29 DE-12 DE-91 DE-BY-TUM DE-19 DE-BY-UBM DE-1049 DE-92 DE-739 DE-898 DE-BY-UBR DE-355 DE-BY-UBR DE-706 DE-20 DE-1102 |
| owner_facet | DE-384 DE-473 DE-BY-UBG DE-703 DE-1051 DE-824 DE-29 DE-12 DE-91 DE-BY-TUM DE-19 DE-BY-UBM DE-1049 DE-92 DE-739 DE-898 DE-BY-UBR DE-355 DE-BY-UBR DE-706 DE-20 DE-1102 |
| physical | XVI, 213 S. Ill., graph. Darst. |
| psigel | ebook |
| publishDate | 2013 |
| publishDateSearch | 2013 |
| publishDateSort | 2013 |
| record_format | marc |
| spelling | Rothenhöfer, Martin 1984- Verfasser (DE-588)104843818X aut Analytical approaches to the study of hyaluronan and hyaluronidases Development and application of hyphenated chromatographic and electrophoretic methods von Martin Rothenhöfer 2013 XVI, 213 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Regensburg, Univ., Diss., 2013 Enzymaktivität (DE-588)4152470-6 gnd rswk-swf Hyaluronsäure (DE-588)4160834-3 gnd rswk-swf Flugzeitmassenspektrometrie (DE-588)4368444-0 gnd rswk-swf Hyaluronidasen (DE-588)4204239-2 gnd rswk-swf Kapillarelektrophorese (DE-588)4290002-5 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Hyaluronsäure (DE-588)4160834-3 s Hyaluronidasen (DE-588)4204239-2 s Enzymaktivität (DE-588)4152470-6 s Kapillarelektrophorese (DE-588)4290002-5 s Flugzeitmassenspektrometrie (DE-588)4368444-0 s DE-604 Erscheint auch als Online-Ausgabe urn:nbn:de:bvb:355-epub-279496 http://epub.uni-regensburg.de/27949/ Verlag kostenfrei Volltext https://nbn-resolving.org/urn:nbn:de:bvb:355-epub-279496 Resolving-System DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=027179986&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Rothenhöfer, Martin 1984- Analytical approaches to the study of hyaluronan and hyaluronidases Development and application of hyphenated chromatographic and electrophoretic methods Enzymaktivität (DE-588)4152470-6 gnd Hyaluronsäure (DE-588)4160834-3 gnd Flugzeitmassenspektrometrie (DE-588)4368444-0 gnd Hyaluronidasen (DE-588)4204239-2 gnd Kapillarelektrophorese (DE-588)4290002-5 gnd |
| subject_GND | (DE-588)4152470-6 (DE-588)4160834-3 (DE-588)4368444-0 (DE-588)4204239-2 (DE-588)4290002-5 (DE-588)4113937-9 |
| title | Analytical approaches to the study of hyaluronan and hyaluronidases Development and application of hyphenated chromatographic and electrophoretic methods |
| title_auth | Analytical approaches to the study of hyaluronan and hyaluronidases Development and application of hyphenated chromatographic and electrophoretic methods |
| title_exact_search | Analytical approaches to the study of hyaluronan and hyaluronidases Development and application of hyphenated chromatographic and electrophoretic methods |
| title_full | Analytical approaches to the study of hyaluronan and hyaluronidases Development and application of hyphenated chromatographic and electrophoretic methods von Martin Rothenhöfer |
| title_fullStr | Analytical approaches to the study of hyaluronan and hyaluronidases Development and application of hyphenated chromatographic and electrophoretic methods von Martin Rothenhöfer |
| title_full_unstemmed | Analytical approaches to the study of hyaluronan and hyaluronidases Development and application of hyphenated chromatographic and electrophoretic methods von Martin Rothenhöfer |
| title_short | Analytical approaches to the study of hyaluronan and hyaluronidases |
| title_sort | analytical approaches to the study of hyaluronan and hyaluronidases development and application of hyphenated chromatographic and electrophoretic methods |
| title_sub | Development and application of hyphenated chromatographic and electrophoretic methods |
| topic | Enzymaktivität (DE-588)4152470-6 gnd Hyaluronsäure (DE-588)4160834-3 gnd Flugzeitmassenspektrometrie (DE-588)4368444-0 gnd Hyaluronidasen (DE-588)4204239-2 gnd Kapillarelektrophorese (DE-588)4290002-5 gnd |
| topic_facet | Enzymaktivität Hyaluronsäure Flugzeitmassenspektrometrie Hyaluronidasen Kapillarelektrophorese Hochschulschrift |
| url | http://epub.uni-regensburg.de/27949/ https://nbn-resolving.org/urn:nbn:de:bvb:355-epub-279496 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=027179986&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT rothenhofermartin analyticalapproachestothestudyofhyaluronanandhyaluronidasesdevelopmentandapplicationofhyphenatedchromatographicandelectrophoreticmethods |