Loyola University Chicago Loyola eCommons Master's Theses Theses and Dissertations 1973 Desmosine and Isodesmosine of Elastin Donald Louis Barbeau Loyola University Chicago Follow this and additional works at: https://ecommons.luc.edu/luc_theses Recommended Citation Barbeau, Donald Louis, "Desmosine and Isodesmosine of Elastin" (1973). Master's Theses. 2654. https://ecommons.luc.edu/luc_theses/2654 This Thesis is brought to you for free and open access by the Theses and Dissertations at Loyola eCommons. It has been accepted for inclusion in Master's Theses by an authorized administrator of Loyola eCommons. For more information, please contact [email protected]. This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License. Copyright © 1973 Donald Louis Barbeau DESMOSINE AND ISODESMOSINE OF ELASTIN BY DONALD LOUIS BARBEAU A mESIS SUBMITTED TO TIIE FACULTY OF TIIE GRADUATE SCHOOL OF LOYOLA UNIVERSITY OF CHICAGO IN PARTIAL FULFILLMENT OF -,THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE JUNE 1973 library • loyola University Medical Center ABSTRACT 11\e determination of desmosine and isodesmosine has recently been employ­ ed to quantitate elastin. Amino acid analysis and high voltage electrophor­ esis, the two methods presently used, are sensitive but time consuming when a large number of samples are to be analyzed. A preliminary investigation of the estimation of these amino acids by colorimetry and fluorimetry, sim­ iliar to the method for hydroxyproline in collagen, is presented. The isolation of desmosine and isodesmosine from elastin hydrolyzates was accomplished efficiently by molecular-exclusion chromatography on Bio­ Gel P-2 followed by ion-exchange chromatography on sulphonated polystyrene 8\ divinylbenzene cross-linked resin. Of many ion exchange resins tried, only Aminex MS "C" resin possessed the proper particle size required for the resolution of these amino acids. Nicotinamide and N(l)methyl nicotinamide were utilized as analogs of _the desmosines for the investigation of possible colorimetric techniques. The reaction of primary aranatic amines with the glutaconic aldehyde, re­ sulting from the destruction of the quaternary pyridinium ring, was stud­ ied. The reaction was sensitive to small concentrations of nicotinamide and the primary aromatic amines. The intensity of the yellow color gave a linear correlation with concentration at 415 to 425 run. The reduction of the pyridiniurn ring of N(l)rnethyl nicotinamide and isodesrnosine with sodium borohydride was performed. N(l)methyl nicotina­ mide exhibited a decrease in absorbance at 264 nm with a simultaneous in­ crease at 358 run. The absorbance of isodesmosine at 269 nm decreased. LIFE Donald Louis Barbeau was born in Chicago, Illinois in 1948. He lived in the Chicagoland area for most of his life and attended St. Joseph High School in Westchester, Illinois. From 1967 to 1971 he attended St. Procopius College in Lisle, Illinois as a Biochemistry major. During his third and fourth years at St. Proco­ pius, he took part in a cooperative research program between the college's Chemistry department and the Swift Research and Development Center in Oak­ brook, Illinois. As a result of the work performed in this program, a thesis and a paper were developed. The thesis fulfilled the partial requirements for the Bachelor of Science Degree at St. Procopius. The paper was presented before the 64th Annual Meeting of the Illinois Academy of Science, Collegiate Chemistry Section, Bradley University (1971); and the Associated Colleges of the Chicago Area (ACCA)'Student Symposium, Chemistry Section, North Cen­ tral College (1971). In the sunnner of 1971, he began his graduate research work at Hines V.A. Hospital in Hines, Illinois under the direction of Dr. A. Dietz and Dr. H. Rubinstein. In the fall of the same year, he entered the Graduate School of Loyola University of Chicago at the Stritch School of Medicine, in the Department of Biochemistry and Biophysics, located in Maywood, Ill­ inois. ACKNOWLEDGEMENTS I would like to express my sincerest appreciation to both Dr. Albert Dietz and Dr. Herbert Rubinstein for supporting my work and educational expenses. I would also like to thank the members of Dr. Dietz's lab­ oratory for their willingness to lend a helping hand when it was needed. TABLE OF CONTENTS . I. INTRODUCTION. PAGE A. ISOLATION AND DETECTION OF DESMOSINE AND ISODESMOSINE. 1 B. COLORIMETRIC AND FLUOROMETRIC REACTIONS OF DESMOSINE ANALOGS. 3 C. OXIDATION AND REDUCTION OF DESMOSINE AND ITS ANALOGS. 4 II. METHODS. A. HYDROLYSIS OF ELASTIN. 6 B. PREPARATION OF BUFFERS FOR COLUMN CHROMATOGRAPHY. 6 c. PAPER CHROMATOGRAPHY OF AMINO ACIDS. 6 -· D. HIGH VOLTAGE ELECTROPHORESIS. 7 E. AMINO ACID ANALYSIS. 8 F. MONITORING EFFLUENT FROM COLUMNS. 8 - - G. ULTRAVIOLET AND VISIBLE ABSORPTION SPECTRA. 9 H. PREPARATION OF N(l)METHYL NICOTINAMIDE. 9 III. EXPERIMENTAL PROCEDURE AND RESULTS. A. FRACTIONATION OF ELASTIN HYDROLYZATES ON SEPHADEX G-15. 10 B. FRACTIONATION OF El.ASTIN HYDROLYZATES ON BIO-GEL P-2. 12 C. ION EXCHANGE CHROMATOGRAPHY. 20 D. AMINO ACID ANALYSIS. 35 -i- IV. COLORIMETRIC AND OPTICAL PROPERTIES OF 1HE DESMOSINES AND THEIR ANALOGS. 43 A. REACTION OF CYANOGEN BROMIDE WITH NICOTINAMIDE. 43 B. pH OPTIMUM FOR REACTIONS OF PRIMARY AROMATIC AMINES WITH THE GLtrrACONIC ALDEHYDE. 44 c. COMPARISON OF SENSITIVITY AMONG THE PRIMARY AROMATIC AMINES. 44 D. ABSORPTION SPECTRA OF POLYMETHINE DYE FORMATION. 51 E. SODIUM BOROHYDRIDE REDUCTION OF N(l)METHYL NICOTrnAHIDE. 51 F. SODIUM BOROHYDRIDE REDUCTION OF ISODESMOSINE. 56 G. REDUCTION OF N(!)METHYL NICOTINAMIDE WITH SODIUM DITHIONITE. 56 v. DISCUSSION. -ii- LIST OF FIGURES FIGURE DESCRIPTION PAGE 1 STRUCTURE OF DESMOSINE AND ISODESMOSINE 2 2 FRACTIONATION OF HYDROLYZATES ON SEPHADEX G-15 11 3 FRACTIONATION OF HYDROLYZATES ON SEPHADEX G-15 13 4 FRACTIONATION OF HYDROLYZATES ON BIO-GEL P-2 16 5 AMINO ACID ANALYSIS OF FRACTION IN ON PA-35 17 6 FRACTIONATION OF HYDROLYZATES ON BIO-GEL P-2 18 7 AMINO ACID ANALYSIS OF FRACTION Io ON PA-35 19 8 FRACTIONATION OF HYDROLYZATES ON Q-15S 22 9 ELUTION OF IN FROM AMINEX MS "C" WITH 0. 20 ! CITRATE pH 4.30 24 -10 ELtn'ION OF Io FROM AMINEX MS "C" WITH 0.20 li CITRATE pH 5.28 26 11 ULTRAVIOLET ABSORPTION SPECTRA OF ISODESMOSINE FROM AMINEX COLUMN 27 12 ELUTION OF Io FROM AMINEX MS "C" WITH 0. 20 li CITRATE pH 4.30 at 56°C 28 13 AMINO ACID ANALYSIS OF DESMOSINE FRACTION (4c) ON PA-35 29 14 AMINO ACID ANALYSIS OF ISODESMOSINE FRACTION (4b) ON PA-35 30 15 ULTRAVIOLET ABSORPTION SPECTRA OF DESMOSINE (4c) AND ISODESMOSINE (4b) 31 16 FRACTIONATION OF HYDROLYZATES ON AMINEX MS "C" WITH 0.20 li CITRATE pH 4.30 AT 56°C 32 -iii- FIGURE DESCRIPTION PAGE 17 ELUTION OF le FRACTIONS FROM AMINEX MS "C" WITii 0.38 !!_ ITRATE pH 4.30 34 18 AMINO ACID ANALYSIS OF EI.ASTIN HYDROLYZATE ON PA-35 36 19 AMINO ACID ANALYSIS OF BASIC AMINO ACID MIXTURE ON PA-35 37 20 AMINO ACID ANALYSIS OF BASIC AMINO ACID MIXT~RE ON PA-35 38 21 AMINO ACID ANALYSIS OF ELASTIN HYDROLYZATE ONAA-15 39 22 PAPER CHROMATOGRAPHY AND HIGH VOLTAGE ELECTRO- PHORESIS OF ELASTIN HYDROLYZATES 42 23 REACTION OF CNBR WITH NICOTINAMIDE 46 24 FORMATION OF POLYMETHINE DYE WITH DESMOSINE AND PRIMARY ARrn.fATIC AMINE 47 25 REACTION OF CNBR WITH NICOTINAMIDE AT THREE TEMPERATURES 48 26 COMPARISON OF SENSITIVITY AMONG VARIOUS PRIMARY AROMATIC AMINE WITH GLUTACONIC ALDEHYDE so 27 ABSORPTION SPECTRA OF £,-PHENYLENEDIAMINE AND CYANOGEN BROMIDE COMPLEX 53 28 ABSORPTION SPECTRA OF E,1J:?.-AMINODIPHENYL AND CYANOGEN BROMIDE COMPLEX 54 29 ABSORPTION SPECTRA OF REDUCED AND NON-REDUCED N(!)METHYL NICOTINAMIDE IODIDE 55 30 ABSORPTION SPECTRA OF REDUCED AND NON-REDUCED ISODESMOSINE 57 -iv- LIST OF TABLES TABLE DESCRIPTION PAGE 1 AMINO ACID COMPOSITION OF SEPHADEX G-15 FRACTIONS 14 2 AMINO ACID COMPOSITION OF BIO-GEL P-2 FRACTIONS 21 3 AMINO ACID ANALYSIS OF ELASTIN HYDROLYZATE ON AA-15 AND PA-35 RESINS 40 4 MOBILITY OF A\f INO ACIDS ON PAPER CHROMATOG- RAPHY AND HIGH VOLTAGE ELECTROPHORESIS 41 5 Sl'RUCTURE OF PRIMARY AROMATIC AMINES 45 6 OPTIMUM pH FOR AROMATIC AMINES FORMING POLYMETHINE DYES 49 7 REDUCTION OF N(!)METHYL NICOTINAMIDE WITH 52 ------ SODIUM BOROHYDRIDE -v- INTRODUCTION During an investigation of the components responsible for the cross­ linking in elastin, two novel amino acids of large molecular weight were discovered by Partridge (1). The two compounds, later designated desmo­ sine and isodesmosine from the Greek 'desmos' meaning to join, are tetra­ aminotetracarboxylic acid isomers containing a pyridinilDll ring as their nucleus (2,3). As the structures indicate in figure 1, the four «-amino carboxylic acid residues present are responsible for the four-fold enhancement of the ninhydrin color yield. This facilitates their detection as amino acids, while their ultraviolet absorption fesulting from the quaternary ring al­ lows them to be characterized more specifically. A complete review of the structure, properties, and early work on the biosynthesis of the desmosines has been compiled (3-5). The isolation of desmosine and isodesmosine from elastin hydrolyzates has mainly been accomplished by elution from sulphonated polystyrene, di­ vinylbenzene crosslinked ion exchange resins (1,6,7). Initially, the desmosines were eluted together from either allDllina or the sulphonated resins. Large volwnes of 1.5 to 4.0 ! HCL are required for the latter technique. Employing the same resin and various concentrations of sodilVll citrate buffers, the desmosines were resolved as discrete ninhydrin and ultraviolet absorbing peaks (1,3). 1be resolution of these isomeric amino acids with citrate buffers has made possible their quantitative detennination and isolation on amino acid analyzers (8). Due to their atypical behavior on these resins, - 1- Desmosine lsodesmosine Figure 1 modifications in buffer and colunm height had to be made (9-17).