ACS Senior Chemists Task Force - Welcome to NESACS enduring has been the Senior ... solutions. He was born on this date. August 12, ... RE-SEED Retirees Enhancing Science Edu-

httpwwwnesacsorg

Summer 2011 Vol LXXXIX No 10

NORTHEASTERN SECTION bull AMERICAN CHEMIC

ALSO

CIET

Y

FOUNDED 1898

NESACS

Summer Scholar ReportBy Kathleen Fleming and Joanne Stubbe

ACS Senior ChemistsTask ForceBy Morton Z Hoffman and Michaeline Chen

National ChemistryWeek Events2011 James FlackNorris AwardTo Peter Mahaffy of Kingrsquos University College

2 The Nucleus Summer 2011

The Nucleus Summer 2011 3

The Nucleus is published monthly except June and August by the Northeastern Section of the AmericanChemical Society Inc Forms close for advertising on the 1st of the month of the preceding issue Textmust be received by the editor six weeks before the date of issueEditor Michael P Filosa PhD ZINK Imaging Inc 16 Crosby Drive Building 4G

Bedford MA 01730 Email Michaelfilosa(at)zinkcom Tel 508-843-9070Associate Editors Myron S Simon 20 Somerset Rd W Newton MA 02465 Tel 617-332-5273

Sheila E Rodman Konarka Technologies Inc 116 John St Suite 12 LowellMA 01852 Email srodman(at)konarkacom tel 978-569-1414 Mindy Levine 516-697-9688 (c) mindylevine(at)gmailcom

Board of Publications Mary Mahaney (Chair) Mindy Levine Vivian K WalworthBusiness Manager Karen Piper 19 Mill Rd Harvard MA 01451 Tel 978-456-8622Advertising Manager Vincent J Gale PO Box 1150 Marshfield MA 02050

Email Manager-vincegale(at)mboservicesnet Tel 781-837-0424Contributing Editors Morton Hoffman Feature Editor Dennis Sardella Book Reviews Calendar Coordinator Sheila Rodman email srodman(at)konarkacomPhotographers Morton Z Hoffman and James PhillipsProofreaders Donald O Rickter Vivian K Walworth Mindy Levine Webmaster Roy HagenCopyright 2011 Northeastern Section of the American Chemical Society Inc

The Northeastern Section of the AmericanChemical Society IncOffice Anna Singer 12 Corcoran RoadBurlington MA 01803 (Voice or FAX) 781-272-1966 e-mail secretary(at)nesacsorgNESACS HomepagehttpwwwNESACSorgOfficers 2011ChairPatrick M Gordon1 Brae CircleWoburn MA 01801Patrickgordon65gmailcomChair-ElectRuth TannerOlney Hall 415BLowell MA 01854University of Mass LowellRuth_Tanner(at)umledu978-934-3662Immediate Past ChairJohn McKewJohnMcKew(at)gmailcomSecretaryMichael SingerSigma-Aldrich3 Strathmore Rd Natick MA 01360774-290-1391 michaelsinger(at)sialcomTreasurerJames Piper19 Mill Rd Harvard MA 01451978-456-3155 piper28(at)attglobalnetAuditorAnthony RosnerArchivistTim FrigoTrusteesPeter C Meltzer Esther A H Hopkins Michael E StremDirectors-at-LargeDavid Harris Stephen Lantos James Phillips Ralph Scannell Myron Simon Alfred ViolaCouncilors Alternate CouncilorsTerm Ends 12312011Doris I Lewis C Jaworek-LopesMary Burgess Patrick M GordonMorton Z Hoffman Lawrence ScottMichael P Filosa Donald RickterKathi Brown Liming ShaoTerm Ends 12312012Amy E Tapper Michaeline F ChenCatherine E Costello Jerry P JasinskiPatricia A Mabrouk Gary R WeismanDorothy J Phillips Marietta SchwartzRuth Tanner Norton P PeetTerm Ends 12312013Thomas R Gilbert Leland L Johnson JrMichael Singer Alfred ViolaRobert Lichter Sophia R SuMary Shultz Kenneth C Mattes

ContentsACS Senior Chemists Task Force __________________________4By Morton Z Hoffman and Michaeline Chen

August Historical Events in Chemistry______________________5By Leopold May Catholic University of America

Announcements________________________________________62011 James Flack Norris Award to Peter Mahaffy NCW 2011Design a T-shirt Contest RE-SEED ndash Retirees Enhancing ScienceEducation through Experiments and Demonstration

Announcements________________________________________7Save the Date-10th Annual Undergraduate Symposium on Sustainability and theEnvironment National Chemistry Week Events

Summer Scholar Report _________________________________8In Vitro Study of Human Ribonucleotide Reductase Enzymatic Activity andAssembly of Diferrictyrosyl Radical CofactorBy Kathleen Fleming and Joanne Stubbe

Cover Participants in the Northeast Student Chemistry Research Conference(NSCRC) (l-r) Michael Lacy (Tufts University) winner of the Excellent PosterPresentation by an Undergraduate Student Award (sponsored by Strem Chemi-cals) April Jewell (Tufts University) Chair NESACS Younger Chemists Com-mittee and organizer of NSCRC Jiazuo Henry Feng (Boston University)winner of the Brauner Memorial Book Award for his oral presentation JenniferBento (Simmons College) recipient of a NESACS Undergraduate Grant-in-Aidwhich provided funds for her to attend and present her poster (in background) atthe ACS National Meeting in Anaheim Photographed at the May EducationNight Meeting (Photo by Morton Z Hoffman)

Deadlines October 2011 Issue August 11 2011November 2011 Issue September 15 2011

All Chairs of standingCommittees the editor of THE NUCLEUS and the Trustees of SectionFunds are members of theBoard of Directors AnyCoun cilor of the American Chemical Soci-ety residing within the section area is an exofficio member of the Board of Directors


The Senior Chemists Task Force(SCTF) was established in 2009 and iscurrently composed of 21 membersIts purpose is to serve as the focalpoint of programming and representa-tion for senior chemists over the age of50 within the ACS and the chemistryenterprise at large Its missionbroadly stated is to encourage and

serve as a conduit for senior membersto volunteer and contribute theirenergy and talent to the ACS includinggovernance education mentoring andcommunity projects to provide usefulservices and information to seniorssuch as retirement and estate planningconsulting and part-time opportunitiesand travelstours to foster networkingopportunities among seniors bothnationally and locally and to representsenior chemists in their interactionwith other elements of ACS gover-nance bringing awareness of theirneeds fostering collaborations andcreating synergies

The age demographics of the ACSdemonstrate the need for institutionalinterest in senior chemists of itsapproximately 160000 members atleast 50 are 50 years of age or olderand about 30 are over 60 SCTF isneeded in order to provide services tothis continually growing segment of

the membership to encourage seniorsto stay involved with ACS to coordi-nate local section activities that involveseniors and to make the rest of theSociety aware of the needs of seniorsFrom a programming standpointSCTF is in a position to organizesponsor and co-sponsor symposia andevents at ACS national meetings andprovide guidance for communicationswith seniors at regional meetings andwithin local sections It can also pro-vide information at its link on the ACSwebsite through the SCTF connectionson the ACS Network and with articlesin local section newsletters the Coun-cilor Bulletin and Committee News

With regard to SCTF program-ming at ACS national meetings themost enduring has been the SeniorChemists Breakfasts which haveattracted sell-out crowds Since 2009the speakers have included PeterStang University of Utah (Salt LakeCity 2009) Luis Echegoyen NSF

Corporate Patrons $2000 - or moreAstraZeneca PharmaceuticalsEisai PharmaceuticalsEMD SeronoGenzyme CorpNovartisJohnson MattheyPfizer IncSchering CorpStrem Chemicals IncVertex Pharmaceuticals

$1000-$1999Boehringer IngelheimGlaxoSmithKlineIrix PharmaceuticalsLyophilization Services of NESundia MeditechYes Bank

$300-$999Cambridge Major LabsGirindusMerrimack ConsultantsOrganixPCI SynthesisSigma AldrichWaters CorpWilmington PharmaTech

ACS SENIOR CHEMISTSTASK FORCEMorton Hoffman and Michaelene Chen SCTF Members

continued on page 12

Tom Beattie (San Diego Local Section) andSCTF member at left with Harry Gray (Cal-Tech) who spoke at the Senior Chemists Break-fast at the ACS meeting in Anaheim March2011 Photo by MZ Hoffman


August 1 1885 One hundred and twenty-six yearsago on this date Georg von Hevesywas born He was a researcher inradioisotopes and discoveredhafnium (Hf 72) in 1923 with DirkCoster In 1943 he was awarded theNobel Prize in Chemistry for hiswork on the use of isotopes as tracersin the study of chemical processes

August 5 1936Robert R Williams and J K Clinesynthesized vitamin B1 on this date

August 6 1960Fifty years ago on this date the firstpublication on the first workinglaser was published in the paperSimulated optical radiation in rubyby Theodore H Maiman in Nature197 494 (1960)

August 8 1779Benjamin Silliman who was bornon this date was a noted teacher atYale University He founded the old-est continuing journal of natural sci-ence in the United States the

American Journal of Science famil-iarly called ldquoSillimanlsquos Journalrdquo In1807 a meteorite fell with spectacu-lar sound and light effects inWeston Connecticut This was thefirst documented fall of a meteoritein the New Worldmdashonly 25 milesfrom New Haven He published ananalysis of the meteorite

August 9 1896Erich Armand Arthur Joseph Huumlckeldeveloped the Huumlckel method ofapproximate molecular orbital (MO)calculations on pi-electron systemsand with Peter Debye developed theDebye-Huumlckel theory of electrolyticsolutions He was born on this date

August 12 1793James Muspratt who was born onthis date improved the methods ofmanufacture of acids and otherchemicals

August 13 1918Frederick Sanger a researcher onthe structure of proteins and insulinand the base sequences of nucleic

acids was born on this date Hereceived the Nobel Prize in Chem-istry in 1958 for his work on thestructure of proteins especially thatof insulin and in 1980 shared thePrize with W Gilbert for their con-tributions concerning the determina-tion of base sequences in nucleicacids and Paul Berg for his funda-mental studies of the biochemistryof nucleic acids with particularregard to recombinant-DNA

August 17 1893Walter K F Noddack co-discoveredrhenium in 1925 with his wife IdaE Noddack and O Berg He wasborn on this date

August 18 1916 Walter J Kauzmann who was bornon this date did research on thehydrophobic effect in the three-dimensional structure of proteinsand the nature of supercooled liq-uids (Kauzmannrsquos paradox)

August Historical Events In Chemistryby Leopold May The Catholic University of America Washington DC 20064

Continued on page 12

RE-SEED Retirees Enhancing Science Edu-cation through Experiments andDemonstrationsSince 1991 the RE-SEED program atNortheastern University has trainedretired scientists and engineers andothers with backgrounds in science ormathematics to provide classroomassistance to K-12 science teachersThere are over eighty volunteers assist-ing science teachers in the greaterBoston area through the Boston RE-SEED Center After taking part in acomprehensive training program par-ticipants typically assist in schoolclassrooms one day a week for oneacademic year The RE-SEED Pro-gram is part of the Center for STEM(science technology engineering andmathematics) Education at Northeast-ern University Other programs focuson science teacher professional devel-opment and student assistance in sci-ence learning

The Boston RE-SEED Center iscurrently recruiting volunteer retiredscientists and engineers for the 2011-2012 school year The recruiting cam-paign is focused on the Boston PublicSchool but volunteers may elect toserve closer to their homes The 32-hour training is being held at theBoston Public Schools Science Centerand will be conducted by the North-eastern University Center for STEMEducation personnel with assistancefrom BPS staff An information meet-ing is being held at Northeastern Uni-versity on August 24 2011 and thetraining will begin September 122011 Call Paul Conroy at 617-737-8388 for more information and to reg-ister for the information session

You can learn more about RE-SEED by visiting their websitewwwreseedneuedu or by calling PaulConroy at 617-373-8388 or by email topaconroyneuedu

The Center for STEM EducationINV520 Northeastern University 360Huntington Avenue Boston MA02115 u


James Flack Norris Award2011 Award to Peter Mahaffy

The Northeastern Section of the Amer-ican Chemical Society is pleased toannounce that Professor Peter Mahaffyis the recipient of the 2011 James FlackNorris Award for Outstanding Achieve-ment in the Teaching of ChemistryWithin the classroom Dr Mahaffy isknown for his highly effective andinnovative teaching methods includinghis commitment to help students edu-cators scientists and the general publicobserve the intricate connectionsbetween science and their everydaylives Dr Mahaffy was instrumental inestablishing and co-directing theKingrsquos Centre for Visualization in Sci-ence which has allowed him to con-tinue his development of digitallearning resources that help learnerssee and understand scientific conceptsthat would otherwise be difficult tovisualize Each month over 10000learners from over 70 countriesadvance their chemical understandingby visiting wwwkeysca where theyaccess information on topics rangingfrom elementary science to chemistryphysics and climate change scienceDuring the International Year of Chem-istry Dr Mahaffy has interacted withand learned from chemists and educa-tors from around the world observingthe imaginative solutions they bring tothe many challenges faced by scien-tists He aspires to build on the IYCthemes and to serve as a catalyst foreducation and understanding thatenables the tools of imagination andscience to make a positive differenceThe Award will be formally presented

to Professor Mahaffy at the November10 meeting of the Northeastern Sec-tion u

National Chemistry Week2011 Design a t-shirt contestWould you like to design the NCW2011 t-shirt worn by all NESACSNCW volunteers The winningdesign will be on the front of thet-shirt The Northeastern Sectionof the American Chemical SocietyLogo and NCW 2011 will be onthe back of the t-shirt Thiscontest is open to all students K-12 in the Northeastern Section Contest rules1 Your design must either capture the

NCW 2011 theme of Chemistry ndashOur Health Our Future or the Inter-national Year of Chemistry Themeof Water Please visit wwwacsorgfor more information

2 You may use up to 4 colors in yourdesign and your design must be onan unlined 85rdquo x 11rdquo sheet ofpaper

3 The deadline for submission is Sep-tember 20 2011 The winner willbe announced by October 1 2011

4 Please mail your original design to Christine Jaworek-Lopes400 The FenwayEmmanuel CollegeBoston MA 02115

5 All entries must have the followinginformation included with theentry studentrsquos name grade homeaddress telephone number schoolname school address teacherrsquosname email and school telephonenumber Both addresses are usedfor sending prizes

6 Have fun u

Announcements

Your one-stop source to career-relatedlinks in the Chemical Sciences

WWWNESACSORGCAREERS


Save theDate10th Anniversary10th Annual UndergraduateSymposium on Sustainabilityand the EnvironmentSaturday November 19 2011Bridgewater State UniversityPlease join us as we celebrate our 10th

anniversary of the only symposiumdedicated to undergraduate environ-mental research and projects thataddress sustainability issues from acampus regional national or globalperspective The event will alsoinclude Phase I tours of our new sci-ence and math center Please email Ed Brush (ebrushbridgewedu) to add your name to ourdistribution list A formal ldquoCall forAbstractsrdquo will be sent electronically inSeptember u

[13] P Chivers K Prehoda R RainesBiochemistry 1997 36 14985-14991

[14] V Pigiet R Conley J Biol Chem1977 252 6367ndash6372

[15] E Artin PhD thesis MIT Cam-bridge MA 2006 Accessed OnlineMIT Dspace

[16] S Salowe J Bollinger J StubbeBiochemistry 1993 32 12749-12760

[17] C Yee M Seyedsayamdost JStubbe Biochemistry 2003 4214541-14552

[18] S Salowe J Stubbe J Bacteriol1986 165 363ndash366

[19] L Thelander J Biol Chem 1973248 4591ndash4601

[20] U Von Dobeln F Eceste Eur JBiochem 1974 43215-220

[21] D Perlstein J Ge J Stubbe Bio-chemistry 2005 44 15366-15377u

Summer Scholar Continued from page 11

National Chemistry WeekEvents

Celebrating ChemistrymdashOur Health Our Future

October 23 2011 ndash Museum of Science Bostonbull Phyllis A Brauner Memorial Lecture by Dr BassamShakhashiriDr Bassam Shakhashiri is a Professor of Chemistry at the University ofWisconsin-Madison and is the William T Evjue Distinguished Chair forthe Wisconsin Idea Professor Shakhashiri has captivated audiences withhis scientific demonstrations at a variety of locations including BostonrsquosMuseum of Science the National Academy of Sciences and the Smith-sonianrsquos National Air and Space Museum in Washington

Taking place in Cahners Theatre (2nd floor Blue Wing) at 100 pmand 400 pm

Admission to the museum is required Free tickets to DrShakhashirirsquos show will be available on a first come first serve basisTickets are available via advance reservation To reserve tickets pleasecontact the NESACS secretary either via email secretarynesacsorg(preferred) or by phone 1-781-272-1966 before October 20 2011 Tick-ets will be available for pick-up in the lobby of the museum at the ACStable

bull Kicking off National Chemistry Week 2011 festivitiesJoin us in a variety of hands-on activities related to the yearly themeTaking place from 100 pm - 500 pm on October 23 2011 throughoutthe Museum

October 29 2011 ndash Boston Childrenrsquos Museum From 11 am ndash 4 pm NCW volunteers will be on-hand throughout themuseum to perform demonstrations and assist in hands-on activitiesrelated to this years theme

September 1 ndash October 21 2011K-12 students participate in the NCW poetry contest Visitwwwnesacsorg andhttpportalacsorgportalacscorgcontent_nfpb=trueamp_pageLabel=PP_TRANSITIONMAINampnode_id=1033ampuse_sec=falseampsec_url_var=region1amp__uuid=c2ba266d-bd00-4469-a4d5-76c2e0eb9d5ffor more infor-mation (after July 15 2011)

June 1 ndash September 20 2011K-12 students participate in the Local Section design a t-shirt competi-tion Visit wwwnesacsorg for more information or see page 6 of thisissue of the NUCLEUS u


0IntroductionRibonucleotide reductases (RNRs) catalyze the conver-

sion of nucleotides to deoxynucleotides in all organisms andplay an essential role in DNA replication and DNA repair[1]

Because of their central role RNRs are also successful tar-gets of several drugs used clinically in the treatment of anumber of malignancies Structurally human RNR (hRNR)consists of two subunits The H1 subunit binds nucleosidediphosphates (NDPs) and the dNTPATP allosteric effectorsThe H2 subunit houses the FeIIIFeIII-tyrosyl radical cofactorrequired to initiate inter-subunit radical propagation (gt35Aring)that leads to thiyl radical generation at the active site on H1to catalyze NDP reduction[12] Mechanism-based inhibitors(MBIs) such as Gemcitabine (Gemzarreg F2C Figure 1a)have been utilized to successfully probe RNR catalyticactivity[3] Clofarabine (Clolarreg ClF Figure 1b) a prodrugindicated for treatment of leukemia is a nucleoside analogproposed to target RNR[4] Elucidating the chemical inacti-vation mechanism of human RNR by Clolarreg is of criticalinterest to further understand both the chemistry of RNRand the clinical efficacy of Clolarreg Prior to studying Clolarregit was first necessary to purify and characterize active hRNRH1 and H2 subunits Unlike E coli RNR much remainsunknown about hRNR specifically the stability of the difer-ric-tyrosyl radical cofactor of human H2 versus E coli β isnot well understood and presents a challenge to conductingin vitro studies Efforts were made to improve protocols forboth the purification of hRNR and the in vitro reassembly ofH2 active cofactor Reported here are (1) improvements tothe purification of H2 (2) a reproducible method for in vitroassembly of the FeIIIFeIII-tyrosyl radical (Ybull) cofactor (3)preliminary stability studies of the Ybull under physiologicalconditions (pH 76 37degC) and (4) an alternative synthesisof Clolar 5rsquondashmonophosphate from Clolarreg Results and DiscussionPurification of H1 and H2 subunits of Human RNRRecombinant human (His)6ndashH1 and (His)6ndashH2 wereexpressed in E coli yielding 12 mgL culture and 38 mgLculture respectively [5] Poor protein yield and purityprompted purification optimization efforts which resulted inimproved yield specific activity and purity[6] Use of Taloncolumn allowed the removal of Arna a 74 kDa E coli pro-tein that previously co-purified with H1[6] Talon was thusused for the purification of (His)6ndashH2 to gt90 homogene-ity as judged by 10 SDSndashPAGE (Figure 2) AsndashisolatedH2 lacks fully active diferric-Ybull cofactor required fornucleotide reduction This cofactor must be assembled invitro following purification H1 was purified by a similarprocedure The specific activity (SA) of H1 (590ndash700

nmolminmg) was measured using [5ndash3H] CDP for the for-mation of 2rsquondashdeoxycytidine 5rsquondashdiphosphate (dCDP) overtime in the presence of the ATP allosteric effector andthioredoxinthioredoxin reductaseNADPH reducing system In vitro Assembly (Reconstitution) of H2 Diferric-Ybull Cofac-tor The inherent instability of asndashisolated mammalian sub-units (hRNR H2 and mouse RNR M2) compared to E coli βpresent a significant challenge to all in vitro experimentsThe in vitro Ybull halfndashlives for E coli RNR β subunit andmouse RNR M2 subunit have previously reported to be onthe order of several days and 10 min respectively[7] Apurification and reconstitution protocol has previously beendeveloped for E coli RNR β subunit that allows study of thestoichiometry and timendashscale of the tyrosine oxidation reac-tion[8] Using this protocol addition of Fe2+ in the presenceof O2 to the purified apo E coli β subunit spontaneouslyleads to assembly of the diferric center and oxidation ofY122 (Equation 1) A modified protocol has recently devel-

Summer Scholar ReportIn vitro Study of Human Ribonucleotide Reductase Enzymatic Activity and Assembly of Diferric-Tyrosyl Radical CofactorKathleen Fleming and JoAnne StubbeDepartment of Chemistry Massachusetts Institute of Technology Cambridge MA 02139

Figure 1 (a) Gemzarreg (F2C) (b) Clolarreg (ClF)

Figure 2 10 SDS PAGE Analysis of Purified H2 (A) and H1 (B)Following Talon Affinity Chromatography Purification The num-bered lanes correspond to (1) Molecular Weight Markers (2) 20microg purified protein (3) 10 microg purified protein (4) 05 microg purifiedprotein


oped for the reconstitution of the H2 active cofactor How-ever the difficulties in reproducibly generating active cofac-tor were noted in initial studies Prior to studying putativemechanism based inhibitors (MBI) of hRNR it is critical toquantify the assembly and stability of the H2 subunit Herecharacterizations of the asndashisolated and reconstituted hRNRH2 subunit are reported

Previous reports of the activity of the reconstituteddiferric-Ybull cofactor of H2 in vitro yielded 08 YbullH2 dimerand SA of 1089 nmolminmg and 75 nmolminmg respec-tively [59] I sought to provide a reproducible method forregenerating (His)6ndashH2 cofactor to the theoretical 10 YbullH2

dimer by first characterizing the asndashisolated H2rsquos iron load-ing tyrosyl radical content and specific activity The ironcontent of the hRNR H2 subunit[5] asndashisolated was meas-ured using a standard ferrozinendashbased colorimetric assayFerrozine binds ferrous iron but not ferric iron and forms acomplex that absorbs strongly at 562 nm (ε562=27870 M-

1cm-1) [10] The specific activity of H2 was measured byradioactive assay and Ybull content per H2 dimer was measuredusing EPR spinndashquantitation and gave 14-16 iron equiva-lents (equiv)dimer 02 YbullH2 dimer and SA of 900ndash1250nmolminmg With this knowledge in hand in vitro recon-stitution was systematically investigated One YbullH2 dimer(Figure 3) following in vitro reconstitution was achievedreproducibly by reducing the amount of iron equivalentsincubated with the H2 in the glovendashbox at 4degC and alterationof the addition rate of Fe2+ to adjust for the asndashisolated pro-tein not being in apo form and to account for potential oblig-atory conformation changes that regulate iron binding Thereconstituted (His)6ndashH2 subunit had 34 iron equivdimer10 YbullH2 dimer and S A of 2100ndash2400 nmolminmg

While working towards developing a reproducible invitro reconstitution method a publication came out report-ing the isolation of H2 (31 iron equivdimer 123 YbullH2dimer and SA of 6000 nmolmingmg) without the in vitroassembly of cofactor[11] These results were obtained byoverexpression of (His)6ndashH2 subunit in E coli harvestingthe cell pellet cell lysate preparation with Bug-buster andBenonase incubation purification with Ni-NTA resin withelution of protein from the resin by gravity dialysis of elu-ate overnight followed by concentration and activity meas-urements Since the half life of the Ybull is 25 min dialysisovernight should leave little radical In my hands the reportfrom this group was irreproducible the (His)6ndashH2 subunitwith 06 iron equivdimer no detectable Ybull and SA of 158nmolminmg Thus we used our optimized protocol

I conducted a preliminary study of the in vitro half-lifeof the H2 Ybull 10 Ybulldimer SA 2100 nmolminmg) by mon-itoring its timendashdependent decay at 37degC and pH 76 Thehalfndashlife was 25 minutes (Figure 4) The instability of thehuman H2 subunit in vitro requires that the decay of Ybull andthe specific activity be monitored during all in vitro inhibi-tion experiments to correct for the spontaneous enzymedecay This in vitro halfndashlife of human H2 contrasts withreported in vitro halfndashlives of E coli and mouse β

Alternative Preparation of ClFMP from ClolarregRecent work noted difficulty purifying ClFMP which wasgenerated enzymatically from Clolarreg and ATP with HdCK[6] This procedure yielded an equilibrium mixture of startingmaterial and products ClFMP and ADP A multindashstepDEAE anion exchange chromatography was utilized to iso-late ClFMP I investigated an alternative twondashstep methodfor the purification of ClFMP from ADP which as foundpreviously coeluted on anion exchange chromatography at350 mM triethylammonium bicarbonate (TEAB) A perio-date cleavage step was introduced to destroy ADP using aprotocol previously reported [12] Sodium periodate selec-

Equation 1 Y-OH + 2Fe2++32O2 +e-+ 3H+ bullO-Y +Fe3+-O-Fe3+ +2 H2O


Figure 3 EPR Spectrum at 20 K of Reconstituted H2 (236 microM)The diferric-tyrosyl radical cofactor of H2 was assembled in vitroEPR spin-quantitation was used to quantify the tyrosyl radicalcontent per H2 dimer EPR parameters 937 GHz microwave frequency 05 mW modulation amplitude 100 kHz modulationfrequency 15 scans DIN 795 DINc=185 YbullH2=[Ybull]std(DINc)sample(DINc)std[H2 dimer]=10 YbullH2 dimer

Figure 4 TimendashDependent in vitro H2 Ybull decay Reconstituted H2(10 YbullH2 dimer) was incubated at 37degC At 0 15 30 45 and 60minute time points 200 μL aliquots were transferred to EPR tubesand immediately flash frozen in liquid N2 for EPR spin-quantita-tion of Ybulldimer


tively reacts with the cisndashdiol of the sugar of ADP to cleavethe 2rsquondashC-3rsquondashC bond generating a dialdehyde The ClFMP isunreactive After removal of excess periodate excess methylamine (pH 75) is added to form iminium ions leading tothe elimination of pyrophosphate Inorganic pyrophos-phatase irreversibly converts the inorganic pyrophosphate toinorganic phosphate Anion exchange chromatography usinga linear gradient from 0ndash600 mM TEAB (pH 68) allowedrecovery of homogenous ClFMP (eluted at 350 mM TEAB) Study of Inhibition of E coli α Subunit by Clofarabine5rsquondashdiphosphate in Presence of 10 Fold Molar Excess β Pre-liminary progress curves for dCDP formation showed possi-ble biphasic time-dependent inhibition These studiessuggested that ClFDP may be a slow-binding reversibleinhibitor of E coli α RNR subunit (Figure 5)Materials and Methods General Clofarabine was pur-chased from AK Scientific The pETndash9d expression vectorfor human deoxycytidine kinase (His6ndashHdCK) was obtainedin E coli BL21 (DE3) pLysS strain as a gift from Dr StaffanEriksson The purification of E coli thioredoxin (TR 40unitsmg) [13] and E coli thioredoxin reductase (TRR 1400unitsmg) [14] HdCK (SA 150 nmolminmg measured byspectrophotometric assay using pyruvate kinase and lactatedehydrogenase) and UMPndashCMP kinase (48 μmolmg minby the [γ-32 P]ATP phosphate transfer assay) have previ-ously been described [15] UVndashvis absorption spectra wereobtained and spectrophotometric assays were carried outusing a Cary 3 UVndashvis spectrophotometer (Varian WalnutCreek CA) Xndashband EPR spectra were acquired using aBruker EMX spectrometer (Bruker Madison WI) Isolation and Characterization of E coli RNR α and β Sub-units Wild type E coli RNR (His)6ndashα subunit (SA 1600-2000 nmolminmg) was purified and prendashreduced aspreviously described [16] protein concentration was deter-mined using Ɛ280nm = 189 mM-1cm-1(6) Wild type E coli(His)6ndashβ subunit was over-expressed in E coli using recom-binant technology and purified from cellular extracts byaffinity chromatography Ni2+ndashNTA resin as previouslyreported [1718] The diferric-tyrosyl-Ybull cofactor was assem-bled in vitro as previously described [17] Protein concentra-tion was determined using Ɛ280nm = 131 mM-1cm-1 [19]

Specific activity (6000ndash7000 nmolminmg) was measuredby radioactive and NADPH coupled spectrophotometricassay Ybull content (1ndash12 radicals per dimer) was measuredboth by the drop-line correction spectroscopy method andby EPR spectroscopy as previously reported [8] EPR spectrawere acquired using a Bruker EMX Xndashband spectrometer at77 K equipped with a quartz finger dewar and at 20 K usingan Oxford Instruments liquid helium cryostat (938 GHzMicrowave Frequency 1 mW Microwave Power 1 GaussModulation Amplitude) Radical content was quantifiedagainst a standard solution of 1 mM CuSO4 in 50 mMEDTA by double integration of spectra registered at non-sat-urating microwave levels by standard Bruker software

Isolation and Characterization of Human RNR H1 and H2Subunits The (His)6ndashH1 and (His)6ndashH2 subunits were puri-fied using a modified protocol reported previously [5] Talon(Clontech) resin was used instead of Ni-NTA and a dATPaffinity column was used as a second step to achieve higherpurity higher specific activies and reduced purification timeIn vitro Assembly of Human RNR H2 Active Cofactor Difer-ric Ybull Human (His)6ndashH2 subunit (50 μM) in 500 μL of 50mM Hepes (pH 76) 100 mM KCl 10 glycerol wasdeoxygenated by six cycles of evaluation (for 3X10 s) fol-lowed by argon flushing using standard Schlenk line tech-nique The deoxygenated (His)6ndashH2 solution was broughtinto the glovendashbox (M Braun Stratham NH) and stored at4degC Incrementally over a 15 min period 3 equivalents of Fe(II) (deoxygenated ferrous ammonium sulfate in 50 mM Tris(pH 76) and 100 mM KCl were added the concentration ofFe(II) was determined by ferrozine assay [10] The resultingmixture was incubated at 4degC for an additional 15 min Theprotein was then removed from the glovendashbox and 170 μL(8-fold excess of 35 equivdimer required) of O2(g) satu-rated buffer was added and O2(g) was blown over the sur-face of the protein solution Excess iron was removed bySephadex G25 chromatography (40 mL 25 X 30 cm) Anactivity assay in the presence of sevenndashfold molar excesshuman (His)6ndashH1 subunit was carried out and 250 μL of theprotein solution was transferred to an EPR tube and frozenin liquid N2 for EPR spinndashquantitation of YbulldimerRadioactive and Spectrophotometric Assays Measurement

of E coli and Human RNR SA The reduction of CDP by Ecoli and human RNR was assayed by measuring the oxida-tion of NADPH coupled to dCDP formation and the forma-


Figure 5 NADPH Coupled Spectrophotometric Inhibition AssayBiphasic Time-Dependent Inhibition of E coli α RNR subunit byClFDP at 25degC (A 0 microM ClFDP B 20 microM C 40 microM D 80microM) The reaction mixture (300 μL 200 μM NADPH 1 mM CDP3 mM ATP 30 μM TR 05 μM TRR 50 mM Hepes (pH 76) 15mM MgCl2 1 mM EDTA 02μM α and 2μM β) without CDP (1mM final concentration saturating substrate conditions) orClFDP (0-80 μM final concentration) was pre-incubated at 25degCfor 1 min CDPCLDP was added and reduction of absorbance at340 nm was continuously monitored for 15 minutes after addi-tion 1 nmol of NADPH oxidized per minute corresponds to 1nmol of dCDP formed per minute


tion of radioactive dCDP from [5ndash3H] CDP In the NADPHoxidation method the disappearance of A340 nm was followedcontinuously using a Cary 3 spectrophotometer (Varian)The following were incubated in a final volume of 300 μL200 μM NADPH 1 mM CDP 3 mM ATP 30 μM TR 05μM TRR 50 mM Hepes (pH 76) 15 mM MgCl2 1 mMEDTA 2 μM (or 02μM) α and 02 μM (or 2μM) β Thereaction mixture was pre-incubated at 25degC for 1 min Thesubunit being assayed in 10-fold molar excess of the othersubunit was added to initiate the reaction Initial velocitieswere measured and used to calculate nmol of NADPH oxi-dized per min 1 nmol of NADPH oxidized per min corre-sponds to 1 nmol of dCDP formed per min [20] For theradioactive assay method a reaction mixture contained in afinal volume of 210 μL 50 mM Hepes (pH 76) 15 mMMgCl2 1 mM EDTA 03 μM (or 3 μM) α 3 μM (or 03μM) β 3 mM ATP 1 mM [5ndash3H] CDP (SA 5926cpmnmolViTrax Placentia CA ) 30 μM E coli TR 05 μMTRR and 1 mM NADPH The assay mixture was pre-incu-bated at 37degC for 2 min and the reaction was initiated bythe addition of [5ndash3H] CDP Aliquots 30 μL each wereremoved over a 10 min time period and quenched in a boil-ing water bath for 2 min dC production was analyzed sub-sequent to dephosphorylation with alkaline phosphatase aspreviously described [21] and analyzed by the method ofSteeper and Steuart [10] The reduction of CDP by hRNR wasmeasured only by the formation of [5ndash3H] dCDP TheNADPH coupled spectrophotometric assay could not beused to measure the specific activity of hRNR subunits orthe holoenzyme since the reduction of absorbance at 340nm versus time is not appreciable compared to the back-ground to allow for specific activity quantitation TimendashDependent Decay of Human H2 Y at 37degC and pH76 Reconstituted human H2 (10 Ybulldimer SA 2100nmolminmg) was incubated at 37degC Aliquots (200 μL)were transferred to EPR tubes at 0 15 30 45 and 60 minand samples were immediately flash frozen in liquid N2 forEPR spin-quantitation of the Ybulldimer Clofarabine to ClFMP The reaction mixture contained in afinal volume of 9 mL 1 mM Clofarabine 5 mM ATP 2 mMDTT 05 mgmL BSA 01 mgmL HdCK 50 mM Tris (pH76) 100 mM KCl and 10 mM MgCl2 The reaction wasinitiated by the addition of hdCK and the mixture incubatedat 37degC for 45 min The reaction mixture was loaded on aDEAE-Sephadex A-25 column (20 mL X 20 cm X 1 cm)equilibrated with 5 mM TEAB (pH 68) and the columnwashed with 50 mL of 5 mM TEAB The product waseluted using a 150 mL X 150 mL linear gradient from 5 to400 mM TEAB Fractions (5 mL) were assayed for A260nmand A280nm the nucleotide containing fractions were com-bined and the solvent was removed in vacuo ClFMP elutedat 350 mM TEAB 31P NMR and 1H NMR revealed contam-inating ADP in addition to the ClFMP product ClFMP waspurified by oxidative cleavage of ADP with periodate fol-

lowed by pyrophosphatase treatment to convert liberatedpyrophosphate into inorganic phosphate as previouslyreported[12] Following this step a second DEAE anionexchange chromatography step with the same aforemen-tioned gradient was used to purify ClFMP ClFMP to ClFDP This synthesis is reported in a publica-tion under review [6]

Spectrophotometric Assay to Study Inhibition of E coli α byClFDP in Presence of 10 Fold Molar Excess β The reac-tion mixture contained in a final volume of 300 μL 200 μMNADPH 1 mM CDP 3 mM ATP 30 μM TR 05 μM TRR50 mM Hepes (pH 76) 15 mM MgCl2 1 mM EDTA 2 μM(or 02μM) α and 02 μM (or 2μM) β The reaction mixturewithout CDP (1 mM final concentration) or ClFDP (0-80μM final concentration) was pre-incubated at 25degC for 1min CDPClDP was added and the reduction of absorbanceat 340 nm was continuously monitored for 15 minutes afteraddition 1 nmol of NADPH oxidized per minute corre-sponds to 1 nmol of dCDP formed per minute [20]

Acknowledgements Thank you to the ACS for supportingme as a recipient of the 2010 James Flack NorrisTheodoreWilliam Richards Summer Research Scholarship Thankyou to Dr Stubbe and members of the Stubbe Laboratoryfor rigorous laboratory training and for sharing their love forbiochemistry researchReferences[1] (a) L Thelander P Reichard Annu Rev Biochem 1979

48133ndash58 (b) P Nordlund P Reichard AnnuRevBiochem 2006 75 681ndash706 (c) J Cotruvo J StubbeAnnu Rev Biochem 2011 80 191ndash1935

[2] J Stubbe DG Nocera CS Yee MC Chang Chem Rev2003 103 2167-2201

[3] (a) E Artin J Wang G Lohman K Yokoyama G YuR Griffin G Bar J Stubbe Biochemistry 2009 4811622-11629 (b) J Wang G Lohman J Stubbe Bio-chemistry 2009 48 11612-11621 (c) J Wang G LohmanJ Stubbe Proc Natl Acad Sci USA 2007 104 14324-14329

[4] (a) P Bonate L Arthaud W Cantrell K Stephenson JSecrist S Weitman NaRevDrug Discovery 2006 5855-863 (b) J Montgomery A Shortnacy-Fowler SClayton J Riordan J Secrist J Med Chem 199235397-401 (c) W Parker S Shaddix C Chang CancerRes 1991 51 2386-2394

[5] J Wang G Lohman J Stubbe Proc Natl Acad SciUSA 2007 104 14324-14329

[6] Y Aye J Stubbe 2010 Submitted[7] M Thelander A Graslund L Thelander J Biol Chem

1985 260 2737ndash2741[8] J Bollinger D Edmondson J Stubbe Science 1991 253

5017 292ndash298[9] O Guittet P HaringkanssonN VoevodskayaS Fridd A

Graumlslund H Arakawa YNakamura L Thelander J BiolChem 2001 276 40647-40651

[10] J Steeper C Steuart Anal Biochem 1970 34 123-130[11] B Zhou Y Yen Mol Cancer Ther 2010 9 1669-1679[12] G Lohman J Stubbe Biochemistry 2010 49 1404-1417


Continued on page 7


(Washington 2009) Robert GrubbsCalTech (San Francisco 2010) RoaldHoffmann Cornell University (Boston2010) Harry Gray CalTech (Anaheim2011) A Senior Chemists Breakfast to be held in Denver on TuesdayAugust 30 will feature Dr BassamShakhashiri current ACS President-Elect as the guest speaker he willspeak on ldquoChemistry and SocietyLooking Back Looking AroundLooking Aheadrdquo

SCTF has organized co-spon-sored or co-listed the following sym-posia on topics important to seniorsand other attendees at the nationalmeetings being a consultant volun-teerism (Washington 2009) the con-sulting business (San Francisco 2010)governmental interface connections toGermany and Europe Medicare sup-plement workshop (Boston 2010)aging and the ACS diverse workforcesin small businesses (Anaheim 2011)In Denver (Fall 2011) SCTF will co-sponsor a symposium on interactionsbetween the Younger Chemists Com-mittee of ACS and the EuropeanYoung Chemists Network ofEuCheMS (European Association forChemical and Molecular Sciences) aswell as symposia on entrepreneurial-ism health care reform and its impacton seniors and the globalization of thechemistry profession

SCTF is in the process of planningfuture activities for seniors includingassistance with consultancies employ-

ment income tax issues and retire-ment and estate planning It antici-pates organizing trips for seniors touniversities for educational visits andto local governmental bodies for leg-islative visits Seniors with academicor industrial backgrounds will becomepart of the ldquoChemistry Ambassadorsrdquoto interact with students and teachers atthe K-12 undergraduate and graduatelevels SCTF plans to work with localsections toward the establishment oftheir own senior chemists committeesfor the promotion of relevant activitiesof interest to their members in theareas of education governmentalaffairs and environmental improve-ment

Later in 2011 the ACS Committeeon Committees (ConC) will evaluatethe programs and activities of SCTFwith an eye toward the establishmentof a national Senior Chemists Commit-tee (SCC) that would be analogous tothe current Younger Chemists Commit-tee (YCC) and Women Chemists Com-mittee (WCC) u

ACS Senior ChemistsContinued from page 4

Members of SCTF at the Senior ChemistsBreakfast at the ACS meeting in AnaheimMarch 2011 left-right George Heinze (NewJersey Local Section) SCTF Chair MortonHoffman (NESACS) Ronald Archer (Connecti-cut Valley Local Section)

Photo by Linda Wang CampEN

D I R E C T O R Y

SERVICES

August 23 1887Bradley Dewey was the ldquoCzarrdquo ofsynthetic rubber production inWorld War II and served as Presi-dent of ACS in 1946 He was bornon this date

August 25 1812 Nicolai N Zinin who was born onthis date discovered the reduction ofaromatic nitro compounds toamines 1842 and the benzidinerearrangement He founded and wasthe first president of the RussianChemical Society 1868-77

August 31 1786Michel E Chevreul was a researcheron dyes and physics of color anddiscovered stearin and margarineHe was born on this date and livedto 100

Additional historical events can befound at Dr Mayrsquos website httpfac-ultycuaedumayChemistrycalendarhtm u

Events in Chemistry Continued from page 5

A catalyst is a little thing that can make a big difference Compact VARIOtrade vacuum systems continuously optimize complex evaporations without programming reducing bumping and tedious oversight

React today Call us to arrange a demo

ProductivityCatalyst

PC3001 VARIOtrade

wwwvacuubrandcom888-882-6730 infovacuubrandnet

Five Decades of Vacuum Innovation


B U S I N E S S D I R E C T O R Y

SERVICESSERVICESSERVICES


CAREER SERVICESSERVICES


SERVICES

Index of AdvertisersBUCHI Corporation 16Boston College 14CreaGen Biosciences 14Eastern Scientific Co16EMD Chenicals Inc 5Front Run OrganX Inc13Huffman Laboratories Inc 13Mass-Vac Inc 4 Micron Inc 13Nacalai USA Inc14New Era Enterprises Inc13Northeastern University 215NuMega Resonance Labs13Organix Inc13PCI Synthesis 12PolyOrg Inc 14Rilas Technologies Inc 13Robertson Microlit Labs 13Vacuubrand Inc12Waters Corporation 14


NO

NPR

OFIT O

RG

U

S POSTA

GE PA

IDN

ORTH

EASTER

N

SECTIO

NA

MER

ICA

N C

HEM

ICA

LSO

CIETY

19 Mill R

oadH

arvard MA

01451

It is now possible to sign up for electronic delivery of theNucleus at wwwnesacsorg You can choose an elec-tronic-only option a paper-only option or receive both anelectronic copy and a paper copy The electronic copy in

general will be available two to three weeks before papercopies delivered by third class mail Improved timelinessshould greatly enhance the value of the Nucleus for ourreaders

If you have any questions contact the editor by email atmichaelfilosa(at)zinkcom u

A Greener NucleusSign up for electronic delivery at wwwnesacsorg

























































6 Have fun u

Announcements


WWWNESACSORGCAREERS


































































Continued on page 7










D I R E C T O R Y

SERVICES









PC3001 VARIOtrade









SERVICES



NO

NPR

OFIT O

RG

U

S POSTA

GE PA

IDN

ORTH

EASTER

N

SECTIO

NA

MER

ICA

N C

HEM

ICA

LSO

CIETY

19 Mill R

oadH

arvard MA

01451




























































6 Have fun u

Announcements


WWWNESACSORGCAREERS


































































Continued on page 7










D I R E C T O R Y

SERVICES









PC3001 VARIOtrade









SERVICES



NO

NPR

OFIT O

RG

U

S POSTA

GE PA

IDN

ORTH

EASTER

N

SECTIO

NA

MER

ICA

N C

HEM

ICA

LSO

CIETY

19 Mill R

oadH

arvard MA

01451













































6 Have fun u

Announcements


WWWNESACSORGCAREERS


































































Continued on page 7










D I R E C T O R Y

SERVICES









PC3001 VARIOtrade









SERVICES



NO

NPR

OFIT O

RG

U

S POSTA

GE PA

IDN

ORTH

EASTER

N

SECTIO

NA

MER

ICA

N C

HEM

ICA

LSO

CIETY

19 Mill R

oadH

arvard MA

01451

































6 Have fun u

Announcements


WWWNESACSORGCAREERS


































































Continued on page 7










D I R E C T O R Y

SERVICES









PC3001 VARIOtrade









SERVICES



NO

NPR

OFIT O

RG

U

S POSTA

GE PA

IDN

ORTH

EASTER

N

SECTIO

NA

MER

ICA

N C

HEM

ICA

LSO

CIETY

19 Mill R

oadH

arvard MA

01451



















6 Have fun u

Announcements


WWWNESACSORGCAREERS


































































Continued on page 7










D I R E C T O R Y

SERVICES









PC3001 VARIOtrade









SERVICES



NO

NPR

OFIT O

RG

U

S POSTA

GE PA

IDN

ORTH

EASTER

N

SECTIO

NA

MER

ICA

N C

HEM

ICA

LSO

CIETY

19 Mill R

oadH

arvard MA

01451






































































Continued on page 7










D I R E C T O R Y

SERVICES









PC3001 VARIOtrade









SERVICES



NO

NPR

OFIT O

RG

U

S POSTA

GE PA

IDN

ORTH

EASTER

N

SECTIO

NA

MER

ICA

N C

HEM

ICA

LSO

CIETY

19 Mill R

oadH

arvard MA

01451
















































Continued on page 7










D I R E C T O R Y

SERVICES









PC3001 VARIOtrade









SERVICES



NO

NPR

OFIT O

RG

U

S POSTA

GE PA

IDN

ORTH

EASTER

N

SECTIO

NA

MER

ICA

N C

HEM

ICA

LSO

CIETY

19 Mill R

oadH

arvard MA

01451








































Continued on page 7










D I R E C T O R Y

SERVICES









PC3001 VARIOtrade









SERVICES



NO

NPR

OFIT O

RG

U

S POSTA

GE PA

IDN

ORTH

EASTER

N

SECTIO

NA

MER

ICA

N C

HEM

ICA

LSO

CIETY

19 Mill R

oadH

arvard MA

01451




























Continued on page 7










D I R E C T O R Y

SERVICES









PC3001 VARIOtrade









SERVICES



NO

NPR

OFIT O

RG

U

S POSTA

GE PA

IDN

ORTH

EASTER

N

SECTIO

NA

MER

ICA

N C

HEM

ICA

LSO

CIETY

19 Mill R

oadH

arvard MA

01451





















Continued on page 7










D I R E C T O R Y

SERVICES









PC3001 VARIOtrade









SERVICES



NO

NPR

OFIT O

RG

U

S POSTA

GE PA

IDN

ORTH

EASTER

N

SECTIO

NA

MER

ICA

N C

HEM

ICA

LSO

CIETY

19 Mill R

oadH

arvard MA

01451














D I R E C T O R Y

SERVICES









PC3001 VARIOtrade









SERVICES



NO

NPR

OFIT O

RG

U

S POSTA

GE PA

IDN

ORTH

EASTER

N

SECTIO

NA

MER

ICA

N C

HEM

ICA

LSO

CIETY

19 Mill R

oadH

arvard MA

01451











SERVICES



NO

NPR

OFIT O

RG

U

S POSTA

GE PA

IDN

ORTH

EASTER

N

SECTIO

NA

MER

ICA

N C

HEM

ICA

LSO

CIETY

19 Mill R

oadH

arvard MA

01451








SERVICES



NO

NPR

OFIT O

RG

U

S POSTA

GE PA

IDN

ORTH

EASTER

N

SECTIO

NA

MER

ICA

N C

HEM

ICA

LSO

CIETY

19 Mill R

oadH

arvard MA

01451






NO

NPR

OFIT O

RG

U

S POSTA

GE PA

IDN

ORTH

EASTER

N

SECTIO

NA

MER

ICA

N C

HEM

ICA

LSO

CIETY

19 Mill R

oadH

arvard MA

01451





NO

NPR

OFIT O

RG

U

S POSTA

GE PA

IDN

ORTH

EASTER

N

SECTIO

NA

MER

ICA

N C

HEM

ICA

LSO

CIETY

19 Mill R

oadH

arvard MA

01451





Documents

ACS Senior Chemists Task Force - Welcome to NESACS enduring has been the Senior ... solutions. He was born on this date. August 12, ... RE-SEED Retirees Enhancing Science Edu-