Copyright is owned by the Author of the thesis. Permission is given for a copy to be downloaded by an individual for the purpose of research and private study only. The thesis may not be reproduced elsewhere without the permission of the Author. DEXTRAN ENZYME IMINE COMPLEXES: A PRELIMINARY STUDY This thesis was presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University Louisa Jane Fisher 1997 11 ABSTRACT A model system involving the formation of protein-dextran complexes has been investigated with a view to improving existing methods of drug administration. Activation of the dextran was achieved by periodate oxidation to give levels of 7%, 21 % and 56% activated glucose moieties. The protein-dextran complexes were investigated with the prospect of obtaining sustained release of proteins from the dextran in an unmodified form. Covalent conjugation of proteins to carbohydrate polymers is known to confer stability on the protein. The proteins in this study were bound to the dextran through imine bonds. The proteins investigated were lysozyme, trypsin, amylase, alcohol dehydrogenase and catalase. The selection covered a range of molecular weights and varying enzymatic activities. As might be predicted, the speed of complex formation was shown to be greater at the 21 % level of activation compared to the 7% activation of dextran in all cases studied. Lysozyme, the smallest protein, readily formed complexes at all three levels of activation. At the 56% level the resulting complex had an extremely high MW, greater than lMDa. The extensive binding between the dextran and lysozyme molecules resulted in a complex that was inactive and showed no signs of releasing any lysozyme, active or inactive. At the lower levels of activation, complex was formed with relative ease. Upon conjugation lysozyme exhibited only minimal activity. Release of a lysozyme-like species with normal lytic activity was observed. Precautions were taken to minimise possible autolysis in the trypsin study. Once complexed it was postulated that autolysis would be prevented or minimised. Similarly the 56% level of activation appeared to be too high to obtain a viable complex for facile trypsin release. Sustained release of a trypsin-like protein was observed with complexes at the 7% and 21 % levels. SEC and SDS-PAGE, in conjunction with a positive BAPNA assay gave support to the released species being trypsin-like. While complexed to the dextran trypsin showed no signs of activity. Released trypsin-like species and unreacted trypsin showed similar tryptic maps from a synthetic peptide, the peptide was designed to show distinctive fragments. a-Amylase, twice the MW of trypsin and over three times the MW oflysozyme, formed complexes with ease at both 7% and 21 % levels of activation. Conjugation to dextran did not effect the activity of a-amylase. Over time the release of an a-amylase-like species from the complex was observed. 111 Alcohol dehydrogenase and catalase are both high MW proteins. Complex formation was observed for each protein. Subsequent experiments showed that upon release the proteins appeared to dissociate, most probably into their subunits. It is also possible that the dimers and monomers bound to the dextran. The main advantage of conjugation in this case appeared to be to confer stability on the proteins. The ADH-complex exhibited enzymatic activity. At 7% and 21 % activation levels the lower MW proteins formed complexes with dextran that exhibited release of a protein species. The higher MW proteins were possibly stabilised when conjugated to dextran, but dissociated upon release. Investigations have shown that the level of activation chosen affects the extent of binding and therefore the functions of the resultant complex. Thus activation levels can be manipulated depending on the desired result. While lower dextran activation levels appeared to be more suited for smaller MW proteins, there were indications that the larger MW proteins could form beneficial complexes at higher activation levels. Results indicated that conjugation to periodate activated dextran could be extended to further proteins with the possibility of therapeutic or commercial applications. iv ACKNOWLEDGEMENTS First and foremost I would like to thank my supervisor Associate Professor David R.K. Harding for his time, input and encouragement over the last two years. I would also like to acknowledge Debbie Frumau for running my amino acid analysis samples, and Dick Poll for his constant help with the FPLC and SMART systems. Thanks are also due to Associate Professor D.N. Pinder and Dr J. Lewis for their time and help with the LLST and Ultracentrifugation experiments respectively. Special thanks and appreciation to Rekha Parshot and Jenny Cross for the SPPS and purification. Thank you also to J. Battersby, Genentech Inc., South San Francisco, for assistance and suggestions with the tryptic digest studies, and for running the HPLC of the trypsin as well as for the gifted rhGH and the rough sketch that lead to Figure 1.8.2. I would also like to thank the Departments of Biochemistry and Chemistry for their assistance along the way, especially the members of the Centre for Separation Science and Gill Norris' s lab. Finally I would like to thank my parents and friends, in particular Suzette, Ruth, Kimberley and Morris for putting up with me especially through the last stages of my thesis. v TABLE OF CONTENTS Abstract ........................................................................................................................ ii Acknowledgements ...................................................................................................... iv Table of Contents .......................................................................................................... v List of Figures ............................................................................................................ viii List of Tables and Schemes ........................................................................................... x List of Abbreviations .................................................................................................... xi CHAPTER ONE INTRODUCTION 1 1.1 Drug Delivery ...................................................................................................... 1 1.2 Controlled Release of Drugs ................................................................................. 2 1.3 Encapsulation ....................................................................................................... 3 1.4 Non-reversible Covalent Bonding ......................................................................... 4 1.5 Sustained Release of rhGH from Dextran ............................................................. 5 1.6 Periodate Oxidation of Dextran ............................................................................ 6 1. 7 Imine Formation ................................................................................................... 9 1.8 Complex Formation of Proteins with Dextran ..................................................... 10 1.9 Protein Modification .......................................................................................... 13 1.10 Investigations into Complex Formation of Proteins to Dextran and Subsequent Release .............................................................................................................. 14 CHAPTER TWO MATERIALS AND METHODS 16 2.1 Reagents and Equipment .................................................................................... 16 2.2 Periodate Oxidation ............................................................................................ 17 2.3 Iodometric Titration ........................................................................................... 17 2.4 Complex Formation ............................................................................................ 17 2.5 Complex Release ................................................................................................ 18 2.6 Complex Reduction Studies ............................................................................... 18 2.7 Lysozytne Lytic Assay ........................................................................................ 18 2. 8 Laser Light Scattering ........................................................................................ 19 2.9 Ultracentrifugation ............................................................................................. 19 2.10 TrypsinBAPNAAssay ....................................................................................... 19 2.11 Trypsin Digest ofrhGH ...................................................................................... 20 2.12 Trypsin Digest of Synthetic Peptide .................................................................... 20 2.13 a-Amylase Activity ............................................................................................ 21 VI 2.14 Alcohol Dehydrogenase Assay .......................... ......................... .... ... ......... ........ 21 2.15 BCA Protein Concentration Determination .................................. ....................... 21 2.16 Amino Acid Analysis Preparation ..... ............................... ... ......... .............. ......... 21 2.17 SDS-polyacrylamide gel electrophoresis ............................................................. 22 CHAPTER THREE LYSOZYME 23 3. 1 Introduction ........................ .. .............................................................. .. ............. 23 3.2 Results and Discussion ............................... .. .. ...... .. .. ............ .. ........ ........ ............ 25 3.3 Conclusions .............. ....... ............... ... ........ ........................... ..... .................. ....... 39 CHAPTER FOUR TRYPSIN 40 4.1 Introduction ..................... ............ ............ ................... ..................................... 40 4.2 Results and Discussion .......................................... ......................................... .. 42 4.3 Conclusions ............................. ... ...................... ....... ............ ............................ 56 CHAPTER FIVE a-AMYLASE 60 5 .1 Introduction ......... .. .......... ...... ........ ... .. ......... ....... .... ........... .. .............................. 60 5.2 Results and Discussion .. .. ..... ... ..... ... .......... .. .. .. ................................................... 62 5.3 Conclusions .. .... ............. ... .. .. .. .... .. ... ........ ...................... .... .. ............................... 71 CHAPTER SIX ALCOHOL DEHYDROGENASE AND CAT ALASE 72 6.1 Introduction ............. .. ............. .. .............................. ........................................... 72 6.1.1 Alcohol Dehydrogenase .................................. ........................................ 72 6.1.2 Catalase ................................................................................... ............... 72 6.1 .3 Higher MW Proteins ........................... .................................................... 73 6.2 Results and Discussion ..................................................................... .................. 74 6.2.1 ADH Complex Formation ................... ...... ............................................ .. 74 6.2.2 Catalase Complex Formation ................ .... .............................................. 74 6.2.3 Complex Formation ....... ...... .............................. ... ........... ....................... 74 6.2.4 ADH-dextran Complex and Release Investigations .................................. 77 6.2.5 Catalase Release ..................................................................................... 85 6.3 Conclusions .... ..... .......... .. ... ....... ... ... ..... .. ...... ... ... .... .... ... ........ ...... .. ... ........ .. ...... .. ... 86 vii CHAPTER SEVEN 88 CONCLUSION AND FUTURE WORK ..................................................................... 88 7 .1 Conclusions ...................................................................................................... 88 7 .2 Future work ..................................................................................................... 90 REFERENCES 93 viii LIST OF FIGURES Figure 1.6.1 Molecular weight distribution by gel filtration of Dextran T-40 6 Figure 1.6.2 Periodate oxidation ofDextran 7 Figure 1.6.3 Overall reaction individual glucose molecule periodate oxidation 8 Figure 1.8.1 Extent of complex formation over increasing dextran activation levels for 24hr period 10 Figure 1.8.2 Possible structure of protein dextran complex 12 Figure 3.1.1 Laser light scattering apparatus 24 Figure 3 .1.2 Diagram of a Schlieren pattern of a homogeneous solution 24 Figure 3.2.1 Complex (ft) formation over time for lysozyme (t) and 56% activated dextran 25 Figure 3 .2.2 Expected progress with time of Schlieren peak 27 Figure 3.2.3 Complex (ft) formation over time between lysozyme (t) and 7% activated dextran 28 Figure 3.2.4 Complex (ft) formation over time between lysozyme (t) and 21 % activated dextran 28 Figure 3.2.5 Release of lysozyme-like (t) species from complex (ft) (lysozyme-21 % activated dextran) over time 29 Figure 3.2.6 SDS-Page 30 Figure 3.2.7 Lysozyme activity 31 Figure 3.2.8 Activity of lysozyme complex with time 32 Figure 3.2.9 Complex formation at 72hrs for reduced and non-reduced complexes 35 Figure 3.2.10 Lytic activity ofreduced and non-reduced complexes 36 Figure 4.1.1 BAPNA assay for trypsin activity 41 Figure 4.2.1 Complex (ft) formation over time between trypsin (t) and 7% activated dextran 43 Figure 4.2.2 Complex (ft) formation over time between trypsin (t) and 21 % activated dextran 43 Figure 4.2.3 Release of trypsin-like species (t) from the complex (ft) 44 Figure 4.2.4 Trypsin activity 45 Figure 4.2.5 Activity oftrypsin-dextran complex over time 47 Figure 4.2.6 Analytical reverse phase chromatography of the released trypsin- like species and the original trypsin 49 Figure 4.2.7 SDS-PAGE analysis 50 Figure 4.2.8 Activity studies on reduced and non-reduced complexes 51 Figure 4.2.9 Reverse-phase analytical run of the synthetic peptide 53 IX Figure 4.2.10 HPLC chromatograph of trypsin digest on the 24mer by the original trypsin. 55 Figure 4.2.11 HPLC chromatograph of trypsin digest on the 24mer by the released trypsin-like species. 56 Figure 5.1.1 Theoretical basis of a-amylase assay procedure 61 Figure 5.2.1 Complex (ft) formation over time between a-amylase (t) and 7% activated dextran 63 Figure 5.2.2 Complex (ft) formation over time between a-amylase (t) and 21 % activated dextran 63 Figure 5.2.3 Release of a-amylase-like species (t) from the dextran complex (ft) over time 64 Figure 5.2.4 Activity of a-amylase 65 Figure 5.2.5 SDS Homogenous gel 66 Figure 5.2.6 Activity of amylase complex over time 67 Figure 5.2.7 Comparison of activities for reduced and non-reduced complexes 70 Figure 6.2.1.1 Complex (ft) formation over time for ADH (t) and 7% activated dextran 75 Figure 6.2.1.2 Complex (ft) formation over time for ADH (t) and 21 % activated dextran 75 Figure 6.2.2.1 Complex (ft) formation over time for catalase (t) and 7% activated dextran 76 Figure 6.2.2.2 Complex (ft) formation over time for Catalase (t) and 21 % activated dextran 76 Figure 6.2.4.1 Complex Formation at 48 Hours between ADH and 7% Activated Dextran 77 Figure 6.2.4.2 Activity assays performed on isolated fractions from ADH-7% dextran complex from figure 6.2.4.1 78 Figure 6.2.4.3 Release from ADH-7% complex (ft) over time 79 Figure 6.2.4.4 ADH Activity 80 Figure 6.2.4.5 SDS- PAGE analysis 82 Figure 6.2.4.6 ADH Reduction studies 84 Figure 6.2.5.1 Release studies for catalase-21 % activated dextran complex (ft) 85 Table 1.10.1 Scheme 3.2.1 Table 3.2.1 Scheme 3.2.2 Table 4.2.1 Table 4.2.2 Scheme 4.2.1 Table 4.2.3 Table 4.2.4 Table 4.2.5 Table 5.2.1 Table 5.2.2 Table 6.2.4.1 Table 6.2.4.2 Scheme 6.2.4.1 LIST OF TABLES AND SCHEMES Molecular weight range of proteins for dextran complex formation study Equilibrium between free protein and dextran Amino acid composition in respect to alanine of the released species in comparison to purified lysozyme and literature sequence Cyano borohydride reduction of protein-dextran complex Specific activity for the trypsin complex samples and the release trypsin-like species Amino acid composition with respect to alanine of the released species in comparison to purified trypsin and the literature sequence Sequence of the 24mer, synthetic peptide AAA of the synthetic peptide AAA composition of peptides from trypsin digest Summary of synthetic peptide digestion Specific activity Amino acid composition of a-amylase and release species Specific activity comparison for ADH-dextran complex Amino acid compositions with respect to alanine Possible reactions occurring with ADH-dextran incubations x 14 32 34 35 46 48 52 53 58 58 67 69 80 81 83 AAA Ab ADH BAPNA BPNPG-7 CD4 DMSO DOR FMOC GI tract GP120 HPLC FPLC LLST met-hGH MWCO NaBHi NaBH3CN NAD+ mPEG PEG PNP rhGH rIGF-1 rIL-2 rtPA SDS-PAGE SEC SPPS Tris TFA TPCK LIST OF ABBREVIATIONS amino acid analysis antibody alcohol dehydrogenase N-a-benzoyl-DL-arginine-p-nitrolanilide HCl blocked p-nitrophenyl maltoheptaoside cell surface glycoprotein receptor for HIV dimethyl sulphoxide double oxidised residues fluorenylmethoxycarbonyl gastro-intestinal tract glycoprotein-120 high performance liquid chromatography fast performance liquid chromatography laser light scattering technique recombinant methionyl human growth hormone molecular weight cut off sodium borohydride sodium cyanoborohydride nicotinamide adenine dinucleotide (oxidised form) monomethoxypoly( ethylene glycol) polyethylene glycol purine nucleoside phosphorylase recombinant human growth hormone recombinant human insulin-like growth factor recombinant human interleukin-2 recombinant human tissue plasminogen activator sodium dodecyl sulphate - polyacrylamide gel electropheresis size exclusion chromatography solid phase peptide synthesis tris-(hydroxymethyl-)aminomethane trifluoroacetic acid L-1-tosylamide-2-phenylethyl chloromethyl ketone X1 Abbreviations used for amino acids: Alanine Arginine Asparagine Aspartic acid Cysteine Glutamic acid Glutamine Glycine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Pro line Serine Threonine Tyrosine Tryptophan Valine Asx Glx Ala Arg Asn Asp Cys Glu Gln Gly His Ile Leu Lys Met Phe Pro Ser Thr Tyr Trp Val asparagine and aspartic acid glutamine and glutamic acid X1l