Biochemistry, Biophysics, and Structural Biology Commons^™

Two-Dimensional Electrophoretic Studies Of Normal Spematozoal Proteins, Ying Lin

Chemistry & Biochemistry Theses & Dissertations

Protein profiles of human spermatozoa have been mapped by using high resolution two-dimensional electrophoresis which combines isoelectric focusing as the first dimension and polyacrylamide gradient gels electrophoresis in SDS as the second dimension. The profiles of normal spermatozoa! extraction, acrosomal extraction and seminal plasma showed high resolution in its separation capability. No qualitative or quantitative differences were observed by using Coomassie Brilliant Blue R-250 staining in a much higher concentration of proteins. However, coomassie blue lacks the sensitivity to detect many minor components. Therefore, a silver staining procedure was used, which resulted in greatly increased sensitivity.

Samples from normal fertile …

A Method For The Comparison Of Hdl Containing Apoprotein E And Hdl Cholesterol, Steven A. Knizner

Chemistry & Biochemistry Theses & Dissertations

Human serum high density lipoproteins (HDL) were isolated by preparative salt density gradient ultracentrifugation and analyzed further by heparin-sepharose affinity chromatography. Separation of the major classes of serum lipoproteins was achieved after a single ultracentrifugation on a salt density gradient at 272,000 x g for 36 hours in a swinging bucket rotor. After isolation, the total HDL was subdivided into HDL containing apoprotein E and HDL without this apoprotein by heparin-sepharose affinity chromatography. Relative amounts of these subclasses were then calculated.

HDL-cholesterol was measured after precipitation of LDL and VLDL by phosphotungstic acid and magnesium. The statistical relationship between HDL …

Investigation Of The Coenzyme Binding Site Of Lactate Dehydrogenase From Chicken Muscle, Jia-Hwei Lin

Chemistry & Biochemistry Theses & Dissertations

Investigation of the coenzyme binding site of lactate dehydrogenase was approached by using coenzyme analogs. Inhibition studies of this enzyme using n-alkylphosphates as coenzyme analogs were performed with a fluorometric method. The results showed that all of the n-alkylphosphates were competitive inhibitors with respect to NAD⁺.

The n-alkyl-ADPs were synthesized from AMP and dicycloheaylammonium salts of n-alkylphosphates. The synthetic compounds were subjected to NMR spectroscopic studies, thin layer chromatography and determination of the ratio of adenosine moiety to phosphate groups. The results indicated that the synthetic compounds were n-alkyl-ADPs.

These synthetic compounds were used as coenzyme analogs in …

High Resolution Two Dimensional Electrophoresis Of Soluble Proteins In Human Spermatozoa, Hsing-Ying Liu

Chemistry & Biochemistry Theses & Dissertations

The evaluation of human semen is very important for diagnosis of male infertility. Combined with the analysis of seminal plasma, a comprehensive biochemical screening of human spermatozoan proteins may be of assistance in providing qualitative and quantitative information to the physician to assist with evaluation.

In this study, human spermatozoa were ruptured by sonication. The soluble spermatozoan proteins were separated from the cellular debris by centrifugation and then analyzed in dissociated forms by two dimensional electrophoresis. The separated proteins were visualized by a silver stain method.

It has been shown that these combined techniques can resolve- four to five hundred …

Determination Of N,N'-Diphenylguanidine In Biological Tissues And Fluids Using High Pressure Liquid Chromatography, Edward Sidney Hunter Iii

Chemistry & Biochemistry Theses & Dissertations

N,N'-Diphenylguanidine (DPG) is a potentially toxic compound used in the vulcanization of rubber products. Distribution of DPG in hybrid mice (C57BL/6J interbred with DAB₂) was determined following intraperitoneal injection. Urine, fecal material, whole blood and tissues were collected at various times after administration of DPG and the extracts analyzed for DPG by high pressure liquid chromatography. Recovery of DPG from spiked urine and whole blood exceed 90%, however, the recovery of DPG from spiked tissue samples was approximately 70%.

DPG concentration in whole blood reached a maximum within ten minutes and rapidly decreased through 180 minutes after administration …

Determination Of Erythrocyte Zinc Concentrations Using Zeeman Effect Atomic Absorption Spectroscopy, Tanya Maranda Lewis

Chemistry & Biochemistry Theses & Dissertations

The purpose of this research project was to develop assay procedures for determination of zinc in erythrocytes. Three methodologies were developed. The methods were evaluated on the basis of within-run and between-run precision. The most reproducible method, the 213.856 nm flame methodology, was used to determine red blood cell zinc concentrations in more than 200 samples obtained from the Neonatal Intensive care Unit at the Children's Hospital of the King's Daughters.

This study demonstrated that erythrocyte zinc levels in premature infants were similar to those of healthy adults. A linear relation between plasma and red blood cell zinc concentrations was …

Investigation Of The Substrate Binding Site Of Nad Glycohydrolase In Human Seminal Plasma, Sau C. Cheung

Chemistry & Biochemistry Theses & Dissertations

Nicotinamide adenine dinucleotide glycohydrolase (NADase) (E.C. 3.2.2.5) from human seminal plasma (HSP) was partially purified through a multistep purification procedure, including affinity chromatography on Amicon Red Matrex Gel A. The NADase activity was assayed by a fluorometric method with nicotinamide 1,N⁶ -ethenoadenine dinucleotide as the substrate. The enzymatic cleavage of etheno-NAD by NADase was accompanied by an eight-fold increase in fluorescence. This large increase in fluorescence provided a sensitive way to follow the rate of etheno-NAD hydrolysis.

NADase was found to be less specific for the purine moiety of NAD, since etheno-NAD was accepted as a substrate. The enzyme …

Development Of An On-Line Computer System For Enzyme Kinetics Studies, Jacqueline R. Thweatt

Chemistry & Biochemistry Theses & Dissertations

The PDP-11/10 minicomputer was interfaced to the Beckman 25 ultraviolet/visible spectrophotometer and this on-line computer system was applied to enzyme kinetics studies. This interface involved hardware equipment to locate, amplify, and transmit the analog signal to an analog-to-digital converter, and software to acquire and store the digital data. Data processing programs were used to calculate kinetic parameters and statistical information from the acquired data.

The Varian Cary 219 ultraviolet/visible spectrophotometer can be readily interfaced to the Apple II Plus microcomputer by hardware equipment which can be purchased from the manufacturer. Various software may also be purchased for use in enzyme …

Isomers Of 5.8s Ribosomal Rna, David Smith

Chemistry & Biochemistry Theses & Dissertations

When whole cell RNA was separated by high resolution PAG (i.e., polyacrylamide gel) electrophoresis, 5.8S rRNA migrated as three higher mobility intense bands, and three faint bands of lower mobility. The three intense bands represent conformational isomers which migrated as a single band under denaturing conditions on PAG. The three faint bands represent conformational isomers of a minor form of 5.8S rRNA which is elongated at the 5' terminus of the molecule. This minor form migrates as a single band of lower mobility than the major form when electrophoresed on a denaturing PAG. The minor form was determined to have …

Development Of A Procedure For Analysis Of High Density Lipoprotein Subclasses, Chan Chin

Chemistry & Biochemistry Theses & Dissertations

Human serum high density lipoprotein subclasses, HDL₂ and HDL₃ , were isolated by preparative salt density gradient ultracentrifugation and further analyzed by electrophoresis on a 4 to 15% concentration gradient polyacrylamide gel.

The separation of the major classes of serum lipoproteins was achieved after a single ultracentrifugation for 272,000 g at 15°c in a swinging bucket rotor. High resolution concentration gradient gel electrophoresis was found to be particularly suitable for the separation of lipoproteins. The isolation of HDL by precipitation methods, heparin-manganese and dextran-magnesium have also been evaluated in this study, and were found not to completely separate …

Electroimmunoassay A New Competitive Protein Binding Assay Using Antibody Selective Electrodes, George Raymond Connell

Chemistry & Biochemistry Theses & Dissertations

The purpose of this study was to utilize concept of antibody selective electrodes to develop a new competitive protein binding assay. This is a new concept and potentially important analytical technique because it combines the advantage of RIA (sensitivity and selectivity) with advantages of electrical assays (speed and low cost).

Electroimmunoassay: A New Competitive Protein Binding Assay Using Antibody Selective Electrodes, George Raymond Connell

Chemistry & Biochemistry Theses & Dissertations

The purpose of this study was to utilize the concept of antibody selective electrodes to developed a new competitive protein binding assay. This is a new concept and a potentially important analytical technique because it combines the advantages of RIA (sensitivity and selectivity) with the advantages of electrical assays (speed and low cost). A conjugate of PGE₂ and dibenzo—18- crown-6 (a cation selective ionophore) was synthesized, and incorporated into a plastic membrane. The ionophore conjugate increased the selectivity of a polyvinyl chloride (PVC) membrane to monovalent cations. Transmembrane potential was altered by anti-PGE₂ antisera in a concentration dependent …

Ortho Cyclization Reactions Of Catecholamines, Robert Lee Hudkins

Chemistry & Biochemistry Theses & Dissertations

A study was undertaken to examine the potential for ortho cyclization associated with the synthesis of 1,2,3,4,-tetrahydroisoquinolines using the Pictet-Spengler reaction. Specif1cally, the condensation of the biogenic amines, dopamine, norepinephrine and epinephrine were carefully examined in light of current literature and the facility and extent of ortho cyclization in each case. The para isomers of these three biogenic amine condensation products were isolated and characterized, while only the ortho isomer derived from dopamine was found stable and could be isolated, and structurally identified. These studies demonstrate that a second cyclization product does, in fact, form in each instance and that …

Correlation Between Glutathione Peroxidase Activity And Selenium Concentrations In Both Plasma And Erythrocytes, Agatha Dudzai Munyanyi

Chemistry & Biochemistry Theses & Dissertations

Plasma and erythrocyte glutathione peroxidase activities and selenium concentrations were determined for 38 apparently healthy female subjects. In addition, erythrocyte lead and plasma lead and iron concentrations were determined in each subject.

Effects of age, smoking, vitamin and mineral supplementation, use of caffeinated beverages and oral contraceptives on glutathione peroxidase activity were determined and interelement relationships were investigated using the one tailed t-test and Spearman Rank Correlation Coefficients

In Vitro Enzymatic Assay Of Rna Methylation, Pamela Jean Eubanks Gallup

Chemistry & Biochemistry Theses & Dissertations

An assay procedure for in vitro enzymatic methylation of mannnalian ribosomal RNA has been developed in this study. The assay procedure, utilized for the comparison of normal and neoplastic methylase activities (using mouse liver and Ehrlich ascites cells as sources of enzyme), is a modification of previously published methods (52,53). A 100,000 x g supernatant (SlOO) enzyme preparation was incubated with 28S-5.8S rRNA and tritium-labeled S-adenosyl-L-methionine. The RNA was extracted, applied to DEAE cellulose paper, washed, and the radioactivity counted. The neoplastic cell methylase preparation was more active in methylating both exogenous neoplastic and normal 28S-5.8S rRNA than the normal …

The Synthesis And Evaluation Of The Biological Activity Of Heterocyclic Analogs Of Phencyclidine, Julian Dew Mckenney Jr.

Chemistry & Biochemistry Theses & Dissertations

Using the classical synthetic approach described by Maddox and co-workers⁴³, the decyanation reaction of 1- piperidinocyclohexane carbonitrile (PCC), 5, was attempted using the heterocyclic anions of 2-methyl-5-nitropyridine, 22, 2-amino-5-nitropyridine, 23, and 2-hydroxy-5-nitropyridine, 24; however, the desired pyridine analogs of phencyclidine (PCP), 1, could not be prepared under these conditions. Attempts to prepare the same analogs via addition of these anions to the iminium tosylate and hydrobromide salts also proved unsuccessful.

The failure to observe the desired decyanation reaction has been attributed to the special sterochemical restrictions of the hindered nitrile of PCC in that facile …

High Performance Liquid Chromatographic Analysis Of Biogenic Amines, Thais Edwina Ahrendt Keegan

Chemistry & Biochemistry Theses & Dissertations

The objectives of this research were to develop a sensitive method for separation and quantitation of biogenic amines and to apply that procedure by monitoring neurochemical changes resulting from stimuli to the test system.

Primary aromatic amines were derivatized with o-phthalaldehyde-mercaptoethanol prior to chromatography, Separation of the fluorescent adduct was achieved using a μBondapak/ phenyl reversed-phase column and a two-step gradient of methanol:0.08 M acetic acid, pH J.O. Fluorescence was monitored using a J40 nm excitation energy and an emission filter which transmits radiation greater than 400 nm (50% T at 418 nanometers).

Following optimization of the chromatographic conditions, studies …

The Synthesis And Biochemical Evaluation Of Potential Acetylcholinesterase Reactivators, Andrea Mcdearmid Lunsford

Chemistry & Biochemistry Theses & Dissertations

Objectives of this research were to (1) synthesize, and (2) evaluate new compounds which would be potential reactivators of organophosphate-inhibited acetylcholinesterase.

A series of 1,8-diazafluorenone and 2,2'bipyridylketone oximes and oxime methiodides were synthesized (11, 13, 14, 22, 24). These compounds were developed as structural analogs of 2-PAM (2-Pyridinealdoxime methiodide) and TMB-4 (1,1Trimethylene-Bis(4-formylpyridinium bromide)dioxime), the current drugs of choice for treatment of organophosphorous poisoning.

Acetylcholinesterase from electric eel was inhibited with DFP (diisopropylfluorophosphate), an irreversible-type inhibitor, and the inhibited enzyme was treated with the various reactivator compounds. The reactivation assays were monitored with pH stat apparatus, and the data were evaluated …

Multiple Forms Of Nicotinamide Adenine Dinucleotide Glycohydrolase From Bull Semen, Stanley D. Yeatts Ii

Chemistry & Biochemistry Theses & Dissertations

Nicotinamide adenine dinucleotide glycohydrolase (NADase) of bull semen is an extracellular, soluble enzyme capable of catalyzing the hydrolysis of the nicotinamide N-ribosidic linkage of NAD⁺. In search for a rapid, simple procedure for the preparation of this enzyme, it was found that Matrex Gel Red A (Amicon) column packing served very well as an affinity gel for NADase. The enzyme was applied to the Matrex Red A column (21 cm x 1.5 cm) in 10 mM sodium phosphate buffer, pH 6.0. Surprisingly, two fractions of NADase activity were obtained when the column was eluted with a sodium chloride …

The Analysis Of S-Adenosylmethionine And S-Adenosylhomocysteine In Normal And Neoplastic Cells, Kathryn Elizabeth Godburn

Chemistry & Biochemistry Theses & Dissertations

Altered patterns of methylation have been shown in both ribosomal and transfer RNA isolated from cancer cells. In contrast to the elevated activity of the methyltransferases responsible for this modification, hypomethylation appears as a general characteristic. The endogenous levels of S-adenosylmethionine and S-adenosylhomocysteine are important in relationship to their role as substrate and product of the transmethylation reaction. The levels of S-adenosylmethionine and S-adenosylhomocysteine were determined using phosphocellulose cation exchange or high pressure liquid chromatography. High voltage paper electrophoresis was used in a modified procedure to directly resolve (³⁵s) labeled S-adenosylmethionine and S-adenosylhomocysteine in small quantities of cell …

High Resolution Two-Dimensional Electrophoretic Study Of Human Seminal Plasma Proteins, Edward E. Gaunt

Chemistry & Biochemistry Theses & Dissertations

A comprehensive biochemical screening procedure for the evaluation of seminal plasma constituents is needed which, when used in conjunction with semen analysis, can provide the physician with both qualitative and quantitative information regarding the fertility status of an individual.

High resolution two-dimensional electrophoresis is a technique whereby proteins in a complex mixture such as a biological fluid are separated in one dimension by molecular charge and in the second dimension by molecular mass. It has been the purpose of this investigation to adapt and evaluate this technique for the analysis of human seminal plasma.

Our efforts have shown that this …

Structural Studies Of Preimplantation Rabbit Embryos Using Ftir Atr, Amy R. Moler-Booth

Chemistry & Biochemistry Theses & Dissertations

FTIR was used in conjunction with a surface selective technique, known as attenuated total reflectance (ATR), to study the infrared spectra of preimplantation rabbit embryos. The rabbit embryos ranged in aged from 3 to 6 days. Since an embryo is mostly water, deuteration of the embryo was necessary to eliminate the intense water background present in the mid-infrared region. A set of spectral standards were tested, in order to identify the bands seen in embryo spectra. By analyzing and comparing the spectra from 3, 4, 5 and 6 day embryos, differences could be noted. In particular, bands defined as Amide …

Sulfatides As Opiate Receptors: An Evaluation Of The Opiate Binding Potential Of Sulfogalactosylgylcerolipid, Darby Geoghegan Hand

Chemistry & Biochemistry Theses & Dissertations

Cerebroside sulfate (CS) has recently been shown to bind opiate drugs in a pharmacologically relevent manner and therefore satisfy the structural requirements of an opiate receptor. Seminolipid (SGG), a novel sulfated galactosyl. glycerolipid that has been isolated from the testes of a variety of mammals, may be viewed as a structural analog of CS. In view of this structural similarity, it is reasonable to presume that SGG may also bind opiate drugs. The isolation of SGG from boar testes was accomplished using silica gel dry column chromotography with a chloroform: methanol water solvent system. The structure and purity of the …

The Association Of 5.8 S With 28s Ribosomal Rna, Nandita Banerjee

Chemistry & Biochemistry Theses & Dissertations

5.8S rRNA is a low molecular weight ribosomal RNA which is noncovalently bonded to the larger ribosomal subunit 28S rRNA; through its 3' end and through its 5' end. This interaction is an integral part of the ribosome, and plays an important role in the ribosome structure and function.

There is a high degree of homology between the 5.8S rRNA primary structures of rat, turtle and chicken. The base sequence of rat 5.8S rRNA differs only in one position from that of turtle and in three positions fr.om that of cl1.icken. Tl1ere is a single purine substitution at the 5' …

Multiple Methods Of Analysis Of 2'-O-Methylation In 5.8s Rrna, Sharon H. Ryan

Chemistry & Biochemistry Theses & Dissertations

5.8S rRNA is a low molecular weight ribosomal RNA which is found hydrogen bonded to 28S rRNA in the eucaryotic cell. There are two nucleotides which have 2'-0-methylations; a guanine residue at position 77 is fully methylated and a uridine residue at position 14 which is partially methylated. This partial 2'-0-methylation of the uridine residue has been found to vary with the tissue source, with the highest level in normal tissue and the lowest level in neoplastic tissue. In order to study the significance of this site-specific methylation, a reliable and convenient method of analysis was needed.

Early studies on …

The Synthesis And Evaluation Of The Potential Antineoplastic Activity Of The Semicarbazone And The Thiosemicarbazone Derivatives Of The Di-2-Pyridyl Ketone And The 1,8-Diazafluorenone Ring Systems, Adriene Y. Principe

Chemistry & Biochemistry Theses & Dissertations

A number of' a-(N)-heterocyclic thiosemicarbazones have been synthesized and evaluated for potential antineoplastic activity since Brockman and colleagues2 reported the antineoplastic activity of' 2-f'ormylpyridine thiosemicarbazone (1). The primary objective of' this research project has been the synthesis and characterization of' the semicarbazone and the thiosemicarbazone derivatives of' the di-2-pyridyl ketone, (9-a) and (9-b), and the 1,8-diazaf'luorenone, (10-a) and (10-b), ring systems, as well as the evaluation of' these compounds for both in vitro and in vivo antineoplastic activity against Ehrlich ascites tumor cells in mice. These studies have suggested that di-2-pyridyl ketone thiosemicarbazone (9-b) is as effective as 2-formylpyridine …

Kinetic And Binding Studies On L-∝-Glycerophosphate Dehydrogenase, Paul Richard Rosevear

Chemistry & Biochemistry Theses & Dissertations

The properties of the coenzyme, NAD, binding site of chicken muscle L-∝-glycerophosphate dehydrogenase were studied. Adenosine monophosphate, adenosine diphosphate and adenosine diphosphoribose were sha.vn to inhibit the enzyme competitively with respect to NAD. The presence of adenosine, pyrophosphate and ribose regions in the coenzyme binding site of the enzyme was suggested by the inhibitor constants obtained for these compounds.

Disodium monoalkyl phosphates, n-butyl to n-dodecyl phosphate, inclusive, were also shown to inhibit the L-∝-glycerophosphate dehydrogenase catalized reaction competitively with respect to NAD. A positive chain length effect was observed in the binding of these compounds to the enzyme, suggesting the …

Studies Of The Purification Of Nad-Dependent And Nadp-Dependent Isocitrate Dehydrogenase By General Ligand Affinity Chromatography, Geoffrey H. Bertkau

Chemistry & Biochemistry Theses & Dissertations

Purification of Yeast NAD⁺-dependent isocitrate dehydrogenase was partially successful when utilizing an NAD⁺-ribosyl-hydrazide Sepharose affinity chromatography column. Although no specific elution system was found, a maximum purification of sixty-five fold was achieved with 0.5M KCl elution. A 38-fold purification was obtained when 20mM NAD⁺ was employed for elution.

Yeast and calf liver NADP⁺-dependent isocitrate dehydrogenase were also found to bind to NAD⁺-ribosyl-hydrazide Sepharose as well as the NAD⁺-dependent enzyme. Again, elution was achieved with either o,5M KCl or 20 mM NADP⁺.

Ammonium sulfate fractionation decreased the yield …

A Batch Method For The Isolation Of Immunogenically Pure Igg, Iga, And Igm From Human Sera, Samuel Christopher Powell

Chemistry & Biochemistry Theses & Dissertations

A method is described for preparing an enriched fraction of the three major antibodies, immunoglobulins IgG, IgA and IgM, from normal human serum by a batch method. Selective precipitation of these proteins was carried out in successive steps using polyethylene glycol (PEG) of average molecular weight 6,000 at concentrations of 7%, 12% and 14% (16) . Zinc sulfate (10 mM) was used to remove highly immunogenic alpha₂ macroglobulin from the IgM fraction. IgA, IgM and IgG were further purified by adsorption methods using DEAE-cellulose. Quantitation and qualitation of the enriched fractions showed nearly complete separation of each of the …