"Nicolae Testemitanu" State University of Medicine and Pharmacy Chair of Biochemistry and Clinical Biochemistry STRUCTURAL BIOCHEMISTRY Practical guide for Faculty of Dentistry students Student _________________________ Group nr. ______________________ Professor ________________________ CHISINAU, 2018
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Ghid Bioch Struct STOM engl 2017-2018...Ninhydrin reaction Method's principle: Ninhydrin reacts with ‐amino groups of amino acids and proteins to form a blue‐violet compound. Procedure:
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Tasksforindividualwork1. Carbon in the unexcited state has the electronic configuration of the outer level
(valencelevel)2s22p2andinthecompoundsitcanhavethedegreeofoxidation+2,but in the excited state it has the electronic configuration 2s12p3. How canschematicallybeshowntheelectrontransition?
Experiment1.PreparationofbuffersolutionsTask.Prepare20mlof0.1mol/laceticbufferwithpH=5.24using0.1mol/lCH3COOHand0.1mol/lCH3COONasolutions.Dissociationconstantofaceticacidis3∙10‐5.Background. Buffer solutions can be prepared in two ways: 1) solutions of eachcomponentsarepreparedseparatelyandafteraremixedinone,2.)onecomponentofthebuffer solution is dissolved in water and another component is added to the obtainedsolution.ItisimportanttoknowthevalueofbuffersolutionpKaandtocalculatesolutionpHusingHenderson–Hasselbalchequation.Procedure1. Calculate howmanymilliliters of 0.1 mol/l CH3COONa and 0.1mol/l CH3COOH are
necessarytomixtoobtain20ml0.1mol/lofaceticbufferwithpH=5.24.2. CalculatetheratioofcomponentsusingtheequationрН=рКа+lg[salt]/[acid].3. Fromtheratioofbuffermixturecomponents results that it shouldcontainXpartsof
saltandYofacid, i.e. all4parts.So, thesaltvolume isequal to (20•x) :4,andacidvolumeisequalto(20•y):4.
Experiment2.Determinationofbloodserumbuffercapacity.Procedure.5mlofbloodserumwithpH=7.4areaddedtotwotest‐tubes.Inonetest‐tubeareadded5dropsofphenolphthaleinandsolutionistitratedwith0.1mol/lNaOHtilltheappearanceof thesamecolorascolorofreferencesolutionthathaspH=9.4. Inanothertest‐tube methyl orange is added and solution is titrated with 0.1 mol/l HCl till theappearanceofthesamecolorascolorofreferencesolutionthathaspH=3.4.
In the conclusion compare the buffer capacity of blood serum by acid and by base andexplainwhythebuffercapacitybyacidisgreaterthanthecapacitybybase.Results:
Conclusion:
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Self‐trainingquestions1. Theoryofsolutions.2. Water–physicalandchemicalproperties,roleinthelivingorganisms.3. Electrolyticdissociationtheory–basicconcepts.4. ThemainconceptsofBronsted‐Lowryprotolytictheoryofacidsandbases.5. Waterdissociation.Theionicproductofwater.6. ThenotionofpH.Solution’spHandpOHmethodsofdetermination.7. The buffer solutions. Principles of buffering. Henderson‐Hasselbalch equation. Buffer
1. Show the composition and themechanism of action of the phosphate buffer systemafteradditionofsmallamountsofstrongacidsorbases.ThepHequationofphosphatebuffer.
2. The content of hydrochloric acid ranges from 0.07 to 0.15% in normal gastric juiceCalculatetherangeofpHchangeignoringtheinteractionforcesbetweentheН+andCl¯ions. C
Experiment №1. Identification of amino acids that contained weak‐bonded sulfur (Folreaction)Method'sprinciple:Weakly bonded sulfur fromproteins and peptides is eliminated byNaOHintheformofNa2S,whichinteractswithNa2PbO2toforminsolublePbS(darkbrownorblackprecipitate).
acids.2. Classification of amino acids according to their chemical structure, physico‐chemical
andacid‐baseproperties.3. Aminoacidsproperties–stereoisomery,solubility,acid‐baseproperties.4. Chemical properties of amino acids – reactions of carboxylation, decarboxylation,
Casestudy:1. Divide the following amino acids in groups according to the biological classification:
Thr,Cys,Phe,Gln,His,Met,Gly,Arg.Writeapeptide that consistsof essential aminoacids.Givethedefinitionof"essentialaminoacid",indicatetheaminoacidsthatbelongto this group and the sources of these amino acids? In which proteins are all theessentialaminoacidspresent?
Experiment1.ChromatographycalidentificationoftheaminoacidsMethodsprinciple: Amino acids have different distributive coefficient in water and inorganic solvent (butanol). Amino acid’s velocity of migration is directly proportional totheirsolubilityinbutanol.Procedure:1.Markthetake‐offlineonthechromatographycalpaperwithapencil.2.Pipetteadropofaminoacidsmixtureinthemiddleofthestartingline(thediameterofthespotmustbelessthen5mm),drythespot.3. Introduce the chromatographycal strip in a vessel with the solvents mixture (water‐butanol).Thestripmustbeinverticalpositionandnottouchthevessel.4.Takethestripoutofthevesselwiththesolventsafter90min,markthedistancepassedbythesolvent(useonlypencil)anddrythechromatogram(10minat70‐100ºC).5.Passthestripthrough0,1‐0,2%ninhydrinsolutionanddryitat100ºC.Onthestripwilldevelopseveralcolouredspots.Eachspotcorrespondtooneaminoacid.6.Measurethefollowingdistances:a –fromthetake‐offlinetothemiddleofeachspot;b –fromthetake‐offlinetothesolvent'sfront.
Conclusion:Whichaminoacidsarepresentinthesolution?__________________________________________________________________________________________________________________________________________________________________________________________________________________Clinical significance: Thismethod allows to determinewhich amino acids and inwhatamountarepresent indifferentbiologicalsamples.Assaysofdifferentbiologicalsamplesfor amino acid's content and composition are indispensable in clinical diagnosis ofnumeroushereditaryerrorsofmetabolism,liver,kidneysdiseasesetc.
Self‐trainingquestions:
1. Protein levels of structural organization: primary, secondary, tertiary and quaternarystructures,generaldescription.Chemicalbondsthatstabilizeeachstructurallevel.Basicnotionsaboutproteinstructuraldomains.
5. Globularproteins:hemoglobin–structureandbiologicrole.6. Fibrillar proteins: collagen and elastin – peculiarities of amino acids composition andstructure.Biologicrole.
7. Ca2+‐binding proteins: clotting factors, Ca2+‐ATPase, calmodulin and collagen.Peculiaritiesoftheaminoacidcompositionthatensurecalciumfixation.Biomedicalrole.
Casestudy:
1.WhatchangeofthehemoglobinstructureischaracteristicforHbSthatcausessicklecellanemia? What repercussions has this change of the primary structure on the upperstructurallevelsoftheprotein,itsfunctionandstateoftheerythrocytes?
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2. Collagen, calmodulin, plasma coagulation factors II,VII, IX andX, etc. areCa2+‐bindingproteins. What is the common structural property of these proteins? What vitaminensuresthisproperty?Whatarethesourcesofthevitamin?Whataretherolesoftheseproteinsinthebody?
Example:each peptide group canformtwohydrogenbonds
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4.What are the similarities and differences of hemoglobin andmyoglobin structure andfunction?What type of hypoxia develops in hemoglobin deficiency?What is the normallevelofhemoglobinintheblood?
a) occursduetoionicbondingoftheadjacentpolypeptidechainsb) hasminimalandmaximalperiodicityc) canbeboth–alpha‐helixandbeta‐structured) appears due to hydrogen bond formation within a single chain and between
Experiment№1:DialysisMethod's principle: Dialysis (fromGreekδιάλυσις,diàlysis, "dissolution"; from διά,dià,"through",andλύσις,lỳsis,"looseningorsplitting")isamethodofseparationonthebasisofmolecularsize.Smallmoleculescanberemovedfromsolutionsbecausetheypassthroughsemipermeablemembrane.Proteinsarelargerthantheporesofthemembraneanddon'tcrossit.Procedure:1. Mixinaflask20mlsolofovalbumineand20dropsofsaturatedsolutionof(NH4)2SO4.2. Putthesolutioninacellophanebagandimmerseditinaglassfullofdistillatedwater.3. After60minpulloutthebagandtransferthesolutionintoatest‐tube.Identifyproteinand(NH4)2SO4inbothsolutions.
4. Thepresenceofproteinsisdeterminedbybiureticreaction(seeThemenr.1).5. The presence of (NH4)2SO4 is determined with BaCl2. To 5 drops of experimentalsolution add 3‐4 drops of 5% sol. BaCl2. Formation of insoluble BaSO4 certified thepresenceofSO42‐.
Self‐trainingquestions:1. Molecularmassoftheproteins.Generalnotionsaboutthemostimportantmethodsfordetermining protein mass – ultracentrifugation, chromatography and massspectroscopy.
2. Amphoteric properties of the protein. The electric charge of the protein. Factors thatdeterminetheelectricchargeoftheprotein.Isoelectricpointandstate.Electrophoresis–principleandbiomedicaluse.Electrophoresisofbloodplasmaproteins.
3. The solubility of the proteins according to the conformation of themolecule and theamino acid composition, solution pH and temperture. Colloidal solutions of proteins.Factorsthatstabilizetheproteincolloidalsolution.Statesofthecolloidalsolutions:sol,gel,xerogel.
4. Denaturation of proteins, agents causing denaturation. Structural changes indenaturatedproteins.Biomedicalrole.
5. Methods of protein separation, purification and analysis: salting, dialysis,electrophoresis and chromatography (ion exchange chromatography, size exclusionchromatography/gelfiltrationchromatographyandaffinitychromatography).Method'sprincipleandbiomedicalimportance.
2. Divide into groups according to their solubility the following proteins: albumin,hemoglobin, keratin, transferrin, IgM, fibrin, prothrombin, collagen. What factorsinfluencethesolubilityoftheseproteins?
4. Asolutioncontainingamixtureofalbuminsandglobulins is treatedwithammoniumsulfate([NH4]2SO4)tosemisaturation,andthentosaturation.Whateffectwillhavethistreatmentonthesolubilityofalbuminsandglobulins?Whatisthemechanismofactionof[NH4]2SO4ontheproteinsfromthesolution?
7. Itisnecessarytoseparateandpurifycertainenzymesfromanimalorplantsourcesfortheproductionofenzymedrugs.Whichofthelistedbelowmethodsisthemostquickand efficient one for the separation and purification of enzymes: denaturation,hydrolysis,electrophoresis,dialysis,affinitychromatography?Describetheprincipleofthemethod.
from the primary structure of a protein?Where is located these sequence in the 3Dstructureof theprotein–on thesurficeof themoleculeor inside?Explain.Write thestructureofthesequence.
b) theelectriccharge,thatdependsonthepHofthesolutionc) electriccharge,thatdependsonthehydrophobicradicalsofaminoacidsd) theaqueousmembrane(MA),whichis formedduetothehydrationofhydrophobicfunctionalgroups
andacid‐baseproperties.13. Chemical properties of amino acids – reactions of carboxylation, decarboxylation,
hydroxylationandtransamination.14. Polypeptide theory of the protein structure. Peptide bond properties. Name and
readingtheaminoacidsinpeptidesandproteins.N‐andC‐terminalaminoacids.15. Methods for determination of amino acid composition and sequence in the
polypeptidechain.16. Levels of structural organizationof the proteinmolecule. Theprimary structure of
the protein. Inheritedmodifications of the primary structure ( sickle cell anemia).Proteinsecondarystructure:types,bondsthatstabilizesecondarystructure.
17. Levelsof structural organizationof theproteinmolecule .The tertiary structureoftheprotein.Typesofintramolecularbondsintheprotein.Quaternarystructureoftheprotein. Cooperative changes of protomers conformation (on the example ofhemoglobinandmyoglobin).Term"domain".
18. Globular and fibrillar proteins (hemoglobin, collagen) ‐ peculiarities ofconformationalandphysico‐chemicalproperties.
20. The biological role of proteins. Albumin, globulins, histones – their features andbiologicalrole.Methodsfordeterminationandseparationofplasmaproteins.
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21. Classesofconjugatedproteins.General featuresofchromoproteins,metaloproteins,nucleoproteins, phosphoproteins, glycoproteins and lipoproteins, their biologicalrole,examples.
22. Physico‐chemicalpropertiesofproteins.The solubilityof theproteins.Factors thatinfluence solubility. Colloidal solutions of proteins, their properties, stabilizingfactors.Saltingout.Dialysisofproteins.
23. Electro‐chemical properties of proteins. Factors determining the charge of theproteins.Isoelectricstateandpoint.Proteinelectrophoresis.
Rezult:________________________________________________________________________________________________________________________________________________________________________________________________________Conclusion:___________________________________________________________________________________________________________________________________________________________________________________________________ C
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Self‐trainingquestions:1. Typesofnucleicacids,functionsandcelllocation.2. Chemicalcompositionofnucleicacids:nitrogenousbases,pentosesandphosphate.3. Nucleosidesandnucleotides–structureandfunctions.4. PrimarystructureofDNA.Polynucleotidechain.Phosphodiesterbonds.5. Secondary and tertiary structure of DNA. DNA double helix – Watson‐Crick model
2.WriteinyournotebookthechemicalstructureofthepolynucleotidesequencedT‐dC‐dG‐dA.Which nucleic acid this sequence belongs to?What will be the electric charge of
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given nucleic acid at physiological pH? What is the role of this electrical charge fornucleosomeformation?
Rezult:________________________________________________________________________________________________________________________________________________________________________________________________________Conclusion:___________________________________________________________________________________________________________________________________________________________________________________________________Experiment№2:FructoseidentificationbySeliwanoff’stestMethod'sprinciple:Seliwanoff’s testisa testwhichseparatesaldoseandketosesugars.Keto sugars are dehydrated by concentrated acids (HCl) to yield furfurals or theirsubsidiaries which react with resorcinol in Seliwanoff reagent to yield a cherry‐redcomplex.Whenaddedtoaldoses,aslowerformingpinkcolorisseen.
Procedure:
№ ReagentsTest‐tubes
I II1 Resorcinpouder 1‐2grains 1‐2grains2 HClconcentr. 2drops 2drops3 Fructose0.5% 2drops 2drops4 Glucose0.5% 2drops 2drops5 Boilfewminutes6 Thecolourofthesolution C
Self‐trainingquestions:1. Thebiologicalroleofcarbohydrates.2. Classificationandstructureofthecarbohydrates.3. Structure and properties of the main monosaccharides (glyceraldehyde,dihydroxyacetone,ribose,deoxyribose,glucose,galactose,fructose).
4. Stereoisomerism of monosaccharides: enantiomers, D‐ and L‐ stereoisomers,diastereolsomersandepimers.
5. Linear and cyclic forms of the monosaccharides. Closed ring structure of 5 or morecarbon atoms (pyranose and furanose rings) monosaccharides. Haworthprojections.The role and properties of the hemiacetal hydroxyl, notions of α‐ and β‐anomers.
6. Important chemical reactions ofmonosaccharides (formation of phosphoric esters,N‐andO‐glycosides,oxidationandreduction).
Experiment №1: Demonstration of the reducing properties of carbohydrates(Fehlingreaction)Method'sprinciple:WhenmonosaccharidesaretreatedwithCu(OH)2inalkalinemedium,abrick‐redprecipitateofCu2Oisformed:
Experiment№1:VitaminC(ascorbicacid)levelassayintheurineMethod's principle: Vitamin C has the property of reducing 2,6‐dichlorophenol‐indophenol(2,6‐DCPIP),whichleadstothechangeinthecolorofthesolution.
Procedure:Nr. Reagents Test-tube 1 Urine 10 ml 2 Н2О dist. 10 ml 3 10% НСl sol. 20 drops 4 Initial color of the solution 5 Titrate with 0,001N 2,6-DCPIP solution 6 Final color of the solution
Clinical significance:Humans cannot make vitamin C (ascorbic acid or ascorbate) andmustobtainitthroughthefoodorsupplements.VitaminCdisappearsfromtheurineearlyinbloodortissuedepletion.Plasmalevelsfallnextandtissuelevels(suchasinleukocytesandplatelets)arethelasttofall.VitaminClevelsinthebodyof1500mgsorlesswillresultin no urinary excretion of vitamin C. However, certain medications such as aspirin,aminopyrine,barbiturates,hydantoinsandparaldehydeaswellascoldorheatstressareknowntoincreasetheexcretionofvitaminCintheurine.Rezult:________________________________________________________________________________________________________________________________________________________________________________________________________Conclusion:___________________________________________________________________________________________________________________________________________________________________________________________________
bondsspecificforthenucleotides.Biologicalroleofnucleotides.3. StructureofDNA–doublehelix.Watson‐Crickmodel.TypesB,AandZofdoublehelix.4. Levels of DNA molecule compaction in prokaryotes (nucleoid) and eukaryotes
9. Stereoisomerism of monosaccharides: enantiomers, D‐ and L‐ stereoisomers,diastereolsomersandepimers.
10. Linear and cyclic formsof themonosaccharides. Closed ring structure of 5 ormorecarbon atoms (pyranose and furanose rings) monosaccharides. Haworthprojections.Theroleandpropertiesofthehemiacetalhydroxyl, notionsofα‐andβ‐anomers.
11. Importantchemicalreactionsofmonosaccharides(formationofphosphoricesters,N‐andO‐glycosides, oxidation and reduction). Ascorbic acid (vitamin C) structure androle.
12. Formationofamino‐carbohydrates(glucosamineandsialicacid),biologicrole.13. Classification of the olygosaccharides – reducing and non‐reducing disaccharides
of phosphatidylehtanolamine that contains linoleic and stearic acids. Show the polarandnon‐polarpartsofthemolecules.
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6. Write the the structures of sphingolipids (ceramide and sphingomyeline) andcerebroside that contain the following fatty acids – palmitic, stearic, oleic andarachidonic.
Experiment№1:EmulsificationcapacityofthebyleacidsMethod'sprinciple:Emulsificationistheprocessofmakinganemulsion,allowingfatandwater tomix, by breaking downof large fat globules into smaller, uniformly distributed
3. Bile acids are synthesized in the liver cells and conjugatedwith glycine and taurineproducing the the corresponding amides. Write the reactions of glycocholic andtaurocholic acids synthesis. What is the biological significance of bile acidsconjugation?
Fatsolublevitamins–A,D,EandKExperiment№1:IdentificationofvitaminsD(Rosenheimreaction)Method's principle: When vitamins D are treated with concentrated trichloroaceticacid(CCl3COOH)aredcompoundisformed.Thecolorofthecompoundturnsblueintime.Procedure:Putintoatest‐tube:
Experiment№2:IdentificationofvitaminsAMethod's principle: When vitamins A are treated with concentrated trichloroaceticacid(CCl3COOH)ayellowcompound is formed.The colorof the compound turnsblue intime.Procedure:Putintoatest‐tube: