Biochemical Assay Biochemistry deals with the identification and quantification of bio-molecules from a variety of living systems Rely on the chemical reactivity and physical properties of bio-molecules to make identification and quantification. Primary tool is the spectrophotometer – Uses absorption of mono chromatic light
Measure quantity Some 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.
Introducing concept of standard curve Uses dilutions of a solution of known concentration to determine concentration of unknown
Standard Curve Assumes that unknown will respond in assay the same as the known – Valid in todays assay as they (the reactive groups. glucose) are the same – Problem in other assay as they may not contain same amount of reactive groups Protein assays (have to choose) But usually close
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
Reducing Sugars Have aldehyde group Can be oxidized to acid Reduces another compound
Requirement placed on sugar Must be an aldehyde – Ketones and hemiacetal configurations are not reducing Conditions of reactions favor conversion to aldehyde by lowering aldehyde concentration
Sugars as Reducing Agents Equilibrium between hemiacetal and open chain is driven to open chain as oxidation to acid form takes place. This ensures a quantitative conversion with time and a stoicheometric production of reduced copper.
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 acid Amount is directly related to [CU +1 ] Will detect any reducing sugar (concentration of sugar must be limiting factor)
3,5-dinitrosalicylic acid (DNS) assay Section A1 pages Sugar reduces the organic DNS which absorbs maximally at yellow wave length Results in change (shift) in absorption spectrum from yellow to red/brown at 540nm – Different from Nelson reaction Measured at 540nm – Unreacted DNS not seen at this wavelength – Amount of absorbance directly related to amount of reducing sugar
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
The DNS assay Experimental design and flow charts page 36 &37 Be sure to read “Hazards” page 37 Protocol on page 38 Data analysis page 42
Today's Experiment Measure 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.
What are we doing today?
Important: See data table page 39 Pipetting technique is critical to accuracy and to preventing cross contamination of samples – Read Micropipette operation (8 to12) – Pipettes have two stops First to take up selected volumes Second to deliver Choose pipette “in the range” that you need.
You will create a standard curve You are provided a stock solution which contains 1.2 mg/ml You 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 concentration You will compare the A540 of unknown to standard curve
Standard curve Uses dilutions of a solution of known concentration to determine concentration of unknown
Protocol Page 38 Steps 8,9,10 – Critical for uniform reaction rates – 100C accelerates the reaction – Cool samples in Ice water bath for 10 to 15 seconds Rapidly brings the sample to low temp which slows the reaction Carefull too long in ice bath will cause condensation on the cuvettes
Important Careful handling of Cuvettes is essential for accuracy and prevent contamination – Handle only with gloves – Touch only the areas not in the light path – Rinse carefully with DH 2 O after each use – Always go from lowest concentration to highest concentration. – Wipe clear surface if necessary with “Kimwipe”
Extremely Important Put cuvette into Spec slot that is in the beam path Be certain that clean panes face the beam path Measure only with the lid closed Always 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 measured – Always use same cuvette
PLEASE DO NOT SLAM THE SPEC LIDS
Important 1. Wear Gloves and Safety Glasses 2. Record the code number of your unknown 3. Be certain that test tubes are clean 4. Water/H 2 O always means distilled water 5.Have TA initial your data before you leave. See lab exit requirements page
Application quiz Address in your report What does the portable glucometers used by diabetics measure? How do they measure it?
Reminder Lab Reports are PERSONAL
Grading for This Experiment Number of lab periods = 1 Lab Report = 10 points Pre lab= 3 points Total = 13 points
Clean up (Please) before you go See page 46. Waste Disposal & Clean up Return pipettes to rack
Next Lab: Enzyme Kinetics Lab C1 Page Read carefully Due next time: Feb:2 & 3. – Prelab assignment for Enzyme Kinetics 1 Lab report for Carbohydrate Analysis – See Report Requirements page 47-48
Cover Page Lab Title Name Date Lab partners Instructor and TA’s
Abstract Theory (background/intro and methods summary) Results
Introduction Conceptual Theory Experimental Theory
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….”
Results Properly labeled data tables and graphs Captions and descriptions Sample calculations (with units!) Other requirements? (Percent error)
Graph Example The 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.9688 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.
Discussion Explain why the experiment was run and what information was gained Answer questions posed in lab manual- look at lab report requirements Results Sources of error