2 Biochemical AssayBiochemistry deals with the identification and quantitation of bio-molecules from a variety of living systemsRely on the chemical reactivity and physical properties of bio-molecules to make Identification and quantitation.Primary tool is the spectrophotometer
4 Measure quantitySome bio-molecules have properties which allow direct measurementproteins have aromatic amino acids (280nm)Nucleic acids have unsaturated ring structures (260nm)Other molecules have chemical properties which can be used in indirect measurement.
5 We will do the DNS assay Section A1 page 4 Measures the reducing capability of glucoseUses a color conversion reaction from yellow to red brown A540Conversion of moles DNS equals moles of glucose.
6 Introduce concept of standard curve Uses dilutions of a solution of known concentration to determine concentration of unknown
7 Standard CurveAssumes that unknown will respond in assay the same as the knownValid in DNS assay as they are the sameProblem in other assay as they may not contain same amount of reactive groupsProtein assays (have to choose)But usually close
8 Reducing Sugars Have aldehyde group Can be oxidized to acid Reduces another compound
9 Requirement placed on sugar Must be an aldehydeKetones and hemiacetal configurations are not reducingConditions of reactions favor conversion to aldehyde
10 Sugars as Reducing Agents Equilibrium betweenhemiacetal and open chainis driven to open chain asoxidation to acid form takesplace. This ensures aquantitative conversion withtime and a stoicheometricproduction of reduced copper.
11 Nelson Assay (a two step Rx) In the Nelson assay Cu+2 is reduced to Cu+1 by the reducing activity of the sugar (step 1)Cu+1 is oxidized to Cu+2 by addition of arsenomolybdic acid (colorless) (step 2)Results in blue (reduced) arsenomolybdous acidAmount is directly related to [CU+1]Will detect any reducing sugar
12 The DNS assay Experimental design page 7 Protocol on page 8 section A4 Data analysis page 10
13 3,5-dinitrosalicylic acid (DNS) Sugar reduces the organic DNS which absorbs maximally at yellow wave lengthResults in change (shift) in absorption spectrum from red/orange to red/brown 540nmDifferent from Nelson reactionMeasured at 540nmUnreacted DNS not seen at this wavelengthAmount of absorbance directly related to amount of reducing sugar
14 The DNS reagent From the MSDS: LABEL PRECAUTIONARY STATEMENTS TOXIC (USA) HARMFUL (EU) HARMFUL BY INHALATION, IN CONTACT WITH SKIN AND IF SWALLOWED. IRRITATING TO EYES, RESPIRATORY SYSTEM AND SKIN. IN CASE OF CONTACT WITH EYES, RINSE IMMEDIATELY WITH PLENTY OF WATER AND SEEK MEDICAL ADVICE.3,5-dinitrosalicylic acid is reduced to 3-amino,5-nitrosalicylic acid
15 Other Assays (page 4 to 7) Mucic Acid test Elson-Morgan test Detects the presence of GalactoseProduces insoluble saccharic acidElson-Morgan testDetects amino-sugars (Hexosamines)Produces a color reactionSeliwanoff testSpecific for ketoseColor reactionBial’s testSpecific for pentose
16 Lab reports for this class (see Report construction Page 12) Abstract. Statements regarding:WHAT you are doing (-> procedure)WHY you are doing it (-> your hypothesis)WHAT you hope to accomplish (-> also hypothesis)Cf. ‘purpose/goal’ in a good lab notebook! Might think of it as a very short introduction.Results/Data/Data AnalysisDiscussion MUST relate data analysis to hypothesis!We chose this format to try to make the workload manageable and quality-oriented.
17 Next week: amino acid titration page 53 Lab B.3 Due next week:Prelab assignment for Titration of an Amino AcidLab report for DNS assayAbstractData table, graph with best-fit line, calculate ave conc of unknown and std dev in average.Discussion: linear relationship? Can also use ‘example questions’ as topics for discussion.
18 Today's ExperimentMeasure the concentration of glucose by detecting the reducing end of the monosaccharide.This group converts the oxidized form of 3,5-dinitrosalicylic acid, DNS, to reduced form which absorbs at 540nm.Amount of reduced DNS proportional to amount of glucose.
19 Important: See data table page 8 Pipetting technique critical to accuracy and to preventing cross contamination of samplesPipetters have two stopsFirst to take up selected volumesSecond to deliverChoose pipetter “in the range” that you need.
20 ImportantCareful handling of Cuvettes essential for accuracy and prevent contaminationHandle only with glovesTouch only the frosted areaRinse carefully with DDH2O after each useAlways go from lowest concentration to highest concentration.Wipe clear surface if necessary with “Kimwipe”
21 Extremely ImportantPut cuvette into Spec slot that is in the beam pathBe certain that clear panes face the beam pathMeasure only with the lid closedAlways set the spec with a blankContains all components of reaction except that which is to be measured
22 Important 1. Wear Gloves and Safety Glasses 2. Record the code number of your unknown3. Be certain that test tubes are clean4. Water/H2O always means distilled water5.Have John or I initial your data before you leave