biochemistry 452 dennis t. brown room 346 polk hall phone 515 5765 e-mail:...

Biochemistry 452

Dennis T. Brown

Room 346 Polk Hall

Phone 515 5765

E-mail: dennis_brown@ncsu.edu Graduate TAs

Section 001: Sayan Chakraborty (schakr10@ncsu.edu)

Section 002: Jigar Desai (jdesai@ncsu.edu)

E-mail

• Major mechanism of communication– Notify you of changes in schedule and project– Send updates on experiment results– Answer your questions

• Keep your mail box clean– Lecture notes on web as too large to send– Some Mails may still push you over quota

Lab Text

• BCH 452 “Experimental Biochemistry, by Hardin-Ascencio-Knopp, Kendall-Hunt Eds. ISBN 978-1-4652-1311-2. Available at the University Bookstore or at Amazon.com (http://www.amazon.com/Experimental-Biochemistry-HARDIN-CHARLES/dp/1465213112/ref=sr_1_1?ie=UTF8&qid=1386083987&sr=8-1&keywords=experimental+biochemistry%2C+hardin)

Lecture Notes and other info posted on Biochemistry web

• http://biochem.ncsu.edu/faculty/brown/brown.php

• At top near photo click on “Biochemistry 452 Notes”

• Look at Powerpoints prior to lab

Schedule

• Lab opens at 1:15– Turn in Pre lab questions & lab reports– Collect any handouts

• 1.30 lecture

• 2:00 lab exercise

• Finish usually around 5:00

NO MAKE UP LABS• Late assignments without a valid excuse will not be accepted and

will receive a score of zero.• Incomplete as a course grade will be awarded only for work not

completed during the course due to conditions deemed by the instructor to be beyond the reasonable control of the student.

• Attendance to all laboratories is mandatory and unexcused absences will affect the outcome of the course. A reduction of 10 points per missed laboratory session will be applied to the final grade for unexcused absences.

Bch 452

• Will introduce fundamental techniques in biochemical analysis

• Will teach the principles upon which the technology depends

• Will make you familiar with theory and practice of the technology

More Importantly BCH 452

• Will stress proper laboratory technique– Reproducibility– Eliminate errors

• Will stress proper record keeping and data reporting– Necessary for archival considerations– For intellectual property– For trouble shooting

Concept of 452• Teach techniques in common use in

biochemistry laboratory science• Try to present situations that exist in a real

laboratory situation– Move from “exercises” with predictable

outcomes.– To “experiments” with doubtful outcomes

• Important to understand and explain anomalous results

My Job

• To be certain that you understand the principles that the experiments will examine

• To be certain that you understand how the apparatus employed in the experiment functions

• To be certain that you understand the reason for each step in the experimental protocol

Lab Safety (Page XXIII)• Students are expected to observe proper laboratory procedures as described in

the lab manual and should sign the disclosure on the first day of lab (Page XXVII)

• a. This is a serious aspect of lab science. Go through the list in the manual.• b. Read each laboratory experiment before you come to class.• c. Follow precautionary statements given in each experiment.• d. Only necessary work materials should be at or on the laboratory bench. Do

no place coats, backpacks, or other personal belongings on your lab bench; store them in the lab cabinets or on the coat rack. Do not place them in the floor.

• e. Do not eat, drink, smoke or place materials in your mouth while in the laboratory.

• f. Wear appropriate clothing and eye protection while in the laboratory and experiments are in progress NO OPEN TOE SHOES

• g. Report all accidents to the instructor or TA immediately: such as spills, cuts, burns, or contact between reagents and your body or eyes. Take appropriate steps in an emergency situation (such as the use of the eye wash station or emergency shower).

Hazardous materials

• Hazardous reagents• Volatiles• Corrosives• Mutagens• Material Safety Data Sheets (MSDS)

– Important information regarding all chemicals– Example on page XXV

Model Molecules

• The three major classes of biomolecules– Carbohydrates– Proteins– Nucleic acids

Model Techniques

• Spectrophotometry

• Centifugation

• Chromatography

• Electrophoresis

• Biochemical fractionation

• PCR

Your Responsibility

• Read the experiment before you come to lab– Read pertaining sections of appendix– Read “Info Boxes”

• Turn in the pre lab exercise• Have all required materials• Some of the protocols can be

confusing. Make a flow sheet that outlines the steps.

Grading

• Lab reports: 130 total points• Pre Lab Questions: 33 points• Exams (2): 25 points each• Total 216 points• A+ 216 - 205 C+ 150 - 140• A 204 - 194 C 139 - 130• A- 193 - 184 C- 129 - 119• B+ 183 - 173 D 118 - 108• B 172 - 162 F 107 - 0• B- 161 - 151

Extra Credit

• 1. An extra 10 points is added to the Protein Purification and Southern blot Lab reports due to increased write-up requirements.

 • 2. Lab C1: 5 points extra credit for Arrhenius plot and

activation energy calculation (see page 94)

 • 3. 7 extra credit points possible for a well-kept lab note

book

Reporting

• Prelab questions– Clearly indicated (see page 28 for next weeks lab)– Due at beginning of designated lab

• Ensures that you have read and understand the exercise

• Completed report– Due at beginning of next exercise

• Report for Lab A1 due at start of lab C1

Laboratory Notebook (ideal) See Page 3

• No loose pages

• Generates copies

• All entries dated

• Each page dated

• Pages signed by a witness attesting to date (extreme) Why??

• Retention time (7 years) Why ??

Lab Reports (Page 4)

• Should contain• Statement of purpose

– Why you are doing experiment– What you wish to determine

• Brief account of theory and design of experiment– Summary or flow chart

• The raw data• All calculations• Results• Conclusions and interpretation

A form for lab write up in lab manual

• Title all sections– Abstract, Discussion, Graph Titles etc.

• Abstract should contain a concise summary of experiment– “I investigated the effect of temperature on activity of

the enzyme Stickase by monitoring its ability to convert stick to broken stick. This was done by determining the accumulation of broken stick by spectrophotometry (A260) at temperatures between 10 to 60 degrees C. I found that Stickase converted stick to broken stick most efficiently at a temperature of 37oC”

Lab Report

• Record all your experimental data including changes in protocol

• Label co ordinates. Be careful with units, spelling and grammar

• Explain sources of error– It is not as important that you get a “correct”

outcome as it is that you can explain your result.

Remember

• Your lab report is your PERSONAL representation of experimental results

• Because you work in groups the raw data produced will be identical however your report should be distinct.

Things that you should know

Units of Biochemistry

• Decade system– Moles– Meters– Liters– Grams– Centigrade

Elementary calculations

• Gram Molecular Weight

• Dilutions

• Rate equations

• “What is the molar concentration of a solution that is 5 millimolar NaCl”

Significant Figures

• Determines the value of a data set

• Includes estimations– 3.2 is not the same as 3.20 or 3.200

Presentation of Data

• Data tables

• Graphs

A Table or Graph Should:

• Be easy to understand

• Project a message

• Does not require extensive reference to text

Experiment

• A solution of compound X is reacted with reagent Y which causes the production of a light absorbing product. The amount of light absorbing product indicates how much compound X is present. The sample containing X is divided into three aliquots and the experiment is repeated three times at three wavelengths.

Lousy Table

Results of the three experiments

Absorbance values for reaction #1 at 360, 420 and 540 nm

Absorbance values for reaction #2 at 360, 420 and 540 nm

Absorbance values for reaction #3 at 360, 420 and 540 nm

0.875 (360) 0.885 (360) 0.823 (360)

0.253 (420) 0.250 (420) 0.244 (420)

0.164 (540) 0.165 (540) 0.157 (540)

Good Table

Absorbance values for the “experiments” at 360,420 and 540 nm

Trial # A360 A420 A540

1 0.876 0.253 0.164

2 0.885 0.250 0.163

3 0.825 0.244 0.157

Bar Graph A: Compares Values at each wavelength, Shows

Reproducible

00.10.20.30.40.50.60.70.80.9

Absorbance

A360 A420 A540

Wavelength

Trial 1Trial 2Trial 3

Bar Graph B: Compares absorbance at different wavelength, shows sensitivity

00.10.20.30.40.50.60.70.80.9

Absorbance

Trial 1 Trial 2 Trial 3

A360A420A540

What is an “Experiment”An example from our research

program

Dr. Raquel Hernandez: Research Associate Professor BiochemistryChief Scientific Officer Arbovax Inc (www.arbovax.com)

Experiment

• Theory

• Hypothesis to test theory

• Experiment suggested by hypothesis

• Design of experiment

• Result of experiment

• Conclusions and expansion of experament

• Translation to practical application

Theory

• Arthropod borne viruses (ArBoViruses)– A class of medically important viruses that are

carried in nature by mosquitos• Dengue virus infects 500million people/year.

1.5billion at risk.• No treatment or cure

• Theory: Arboviruses are insect viruses which have evolved over millions of years to infect vertibrates

Hypothesis

• As they evolved to infect vertebrates, Arboviruses acquired new genetic information necessary for growth in this completely different genetic and biochemical environment

• It should be possible to identify those genetic elements and remove them– Produce virus which can grow in insects but

not vertebrates

Virus Structure as a Target

Outer shell, 2 proteins

Membrane bilayer. Is penetratedby the two proteins in the outer shell

Core. Contains multiple copies ofone protein surrounding RNA

Sindbis Virus Envelopment

E2-E1 Trimer

Sindbis Virus Glycoproteins

E2-Capsid Binding

Domain

Systematically removeamino acids fromtransmembrane domain

Mammalian and insect membranes

• Insect membranes have no cholesterol

• Cholesterol makes membranes viscous, ion impermeable and thick

• Insect membranes are 4 nm thinner

• Membrane protein must span both insect and mammalian membrane

Wild Type Deletion Mutant

E2 Transmembrane MutantsVYTILAVASATVAMMIGVTVAVLCAC Wild Type (TM26) VYTILAVASATVAMMIGVTVAVLCAC TM25(D379M)VYTILAVASATVAMMIGVTVAVLCAC TM24VYTILAVASATVAMMIGVTVAVLCAC TM23VYTILAVASATVAMMIGVTVAVLCAC TM22VYTILAVASATVAMMIGVTVAVLCAC TM21VYTILAVASATVAMMIGVTVAVLCAC TM20VYTILAVASATVAMMIGVTVAVLCAC TM19VYTILAVASATVAMMIGVTVAVLCAC TM18VYTILAVASATVAMMIGVTVAVLCAC TM17VYTILAVASATVAMMIGVTVAVLCAC TM16VYTILAVASATVAMMIGVTVAVLCAC TM14VYTILAVASATVAMMIGVTVAVLCAC TM12VYTILAVASATVAMMIGVTVAVLCAC TM10VYTILAVASATVAMMIGVTVAVLCAC TM8

Hernandez et al. J Virol. 2003 Dec;77(23):12710-9.

TM16 & TM17 are host restricted mutants

1.00E+001.00E+011.00E+021.00E+031.00E+041.00E+051.00E+061.00E+071.00E+081.00E+091.00E+10

Mutant

Growth of TM mutants in mammalian and insect cells

Conclusions

• Shortening transmembrane domain crates a host range (HR) mutation– Restricts efficient growth to insect cells

• Suggests approach for making vaccines for arboviruses– Use HR mutants as vaccines

VIRUS DOSE MORTALITY MORBIDITY CHALLENGE MORBITITYPOST CHALLANGE

MORTALITYPOST CHALLENGE

1 Mock(buffer)

NA 0% 0% SAAR86 1000 pfu i.c.

92 % 36%

2 TM16 103 0% 0% SAAR86 1000 pfu i.c.

68% 48%

3 TM16 UV NA 0% 0% SAAR86 1000 pfu i.c.

92% 36%

4 TM17 103 0% 0% SAAR86 1000 pfu i.c.

0 0

5 TM17 UV NA 0% 0% SAAR86 1000 pfu i.c.

84% 36%

Vaccination of mice with TM16 & 17

Patent StrategyUS Patent No. 6,589,533Issued July 8, 2003"Genetically-engineered alphaviruses, flaviviruses, and bunyaviruses with modified envelope transmembrane glycoproteins and altered host-range phenotype"

 US Patent No. 6,306, 401Issued October 23, 2001"Genetically-engineered sindbis virus with modified E2 Glycoprotein and altered host-range phenotype"

US Patent No. 7,335,363Issued February 2008"Membrane Virus Host-Range Mutations and their use as Vaccine Substrates"

Three other patents pending

License Patents

Malcolm Thomas, Chief Executive OfficerDr. Raquel Hernandez, Chief Scientific Officer

Arbovax Products

• Vaccine for Dengue Fever– Developed for all 4 serotypes– Very successful non humane primate trial

• 100% Sero-conversion to all 4 serotypes– Ready for phase one human clinical trials

• Approved by Food and Drug Administration

• Vaccine for Chikungunya Fever– Successful trials in mice– Ready for non human primate trials

• Vaccine for West Nile Fever– Host range mutants made– Ready for trials in mice

Translational Science

• From Theory (basic Science) to Medical Product (application)

• Strategy will work for any of the 700 known arboviruses– 150 are known to cause human disease– Some cause disease in animals of agricultural

importance

Next Exercise

• Carbohydrates and standard curve– Section A1 in lab manual (page 26)– Read carefully prior to coming to lab.– Do the pre lab questions turn them in on the

26th-27th

Benchtop Protocols

• Page 387

• The benchtop protocol summarizes step be step the days experiment. It is more convenient than having the entire manual open on your bench

DVD

• The DVD shows step by step each part of the experimental protocol, watching it will help prepare you for the days exercise