2 Biochemical AssayBiochemistry deals with the identification and quantification of bio-molecules from a variety of living systemsRely on the chemical reactivity and physical properties of bio-molecules to make identification and quantification.Primary tool is the spectrophotometerUses absorption of mono chromatic light
4 Measure quantitySome bio-molecules have properties which allow direct measurement.proteins 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 Introducing concept of standard curve Uses dilutions of a solution of known concentration to determine concentration of unknown
6 Standard CurveAssumes that unknown will respond in assay the same as the knownValid in todays assay as they (the reactive groups. glucose) are the sameProblem in other assay as they may not contain same amount of reactive groupsProtein assays (have to choose)But usually close
7 Our model carbohydrate is the sugar glucose We will exploit its ability to reduce other compounds to produce a product which can be measured optically
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 by lowering aldehyde concentration
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 (concentration of sugar must be limiting factor)
12 3,5-dinitrosalicylic acid (DNS) assay Section A1 pages 33-49 Sugar reduces the organic DNS which absorbs maximally at yellow wave lengthResults in change (shift) in absorption spectrum from yellow to red/brown at 540nmDifferent from Nelson reactionMeasured at 540nmUnreacted DNS not seen at this wavelengthAmount of absorbance directly related to amount of reducing sugar
13 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
14 The DNS assay Experimental design and flow charts page 36 &37 Be sure to read “Hazards” page 37Protocol on page 38Data analysis page 42
15 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.
17 Important: See data table page 39 Pipetting technique is critical to accuracy and to preventing cross contamination of samplesRead Micropipette operation (8 to12)Pipettes have two stopsFirst to take up selected volumesSecond to deliverChoose pipette “in the range” that you need.
18 You will create a standard curve You are provided a stock solution which contains 1.2 mg/mlYou will dilute this stock solution in a specified manner always producing a 4 ml solution (See table A1-2)After reacting with DNS you will read the absorbance of each solution at 540 and plot vs concentrationYou will compare the A540 of unknown to standard curve
19 Standard curveUses dilutions of a solution of known concentration to determine concentration of unknown
20 Protocol Page 38 Steps 8,9,10 Critical for uniform reaction rates 100C accelerates the reactionCool samples in Ice water bath for 10 to 15 secondsRapidly brings the sample to low temp which slows the reactionCarefull too long in ice bath will cause condensation on the cuvettes
21 ImportantCareful handling of Cuvettes is essential for accuracy and prevent contaminationHandle only with glovesTouch only the areas not in the light pathRinse carefully with DH2O after each useAlways go from lowest concentration to highest concentration.Wipe clear surface if necessary with “Kimwipe”
22 Extremely ImportantPut cuvette into Spec slot that is in the beam pathBe certain that clean panes face the beam pathMeasure only with the lid closedAlways set the spec with a blank (line 1 table A.1-2, page 39)Contains all components of reaction except that which is to be measuredAlways use same cuvette
24 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 TA initial your data before you leave. See lab exit requirements page
25 Application quiz Address in your report What does the portable glucometers used by diabetics measure?How do they measure it?
32 Cover PageLab TitleNameDateLab partnersInstructor and TA’s
33 AbstractTheory (background/intro and methods summary)Results
34 IntroductionConceptual TheoryExperimental Theory
35 Methods Protocol with general description “In a beaker, 5ml of reagent X was mixed with 2ml of reagent Y…”“1) Obtain gloves, lab coat, four micropipettes and a clean beaker . 2) Set a micropipette to 1000μl….”
36 Results Properly labeled data tables and graphs Captions and descriptionsSample calculations (with units!)Other requirements? (Percent error)
37 Graph ExampleThe following graph shows standard curve of glucose concentration. Absorbency readings were taken at 540 nanometers of 5 samples with known glucose concentration. R2 value of indicates a fit linear correlation. The slope of this graph was used to calculate glucose concentration in unknown samples (Fig 4).Fig 3: Graph of concentration of “standard” glucose vs. absorbancy at 540 nm for tubes 1-5.
38 DiscussionExplain why the experiment was run and what information was gainedAnswer questions posed in lab manual- look at lab report requirementsResultsSources of error