text:
An improvement in the process of sequentially degrading peptide chains in a reaction vessel by successive coupling and cleavage reaction steps, wherein the vessel is subjected to a vacuum purging operation between the reaction steps to remove undesired materials. The improvement involves flushing the vessel with a stream of nitrogen gas while it is under vacuum whereby the undesired materials are more rapidly and more completely removed from the vessel and the cumulative yield of the process is increased.
Claims available in
Description (OCR text may contain errors)
United States Patent [1 1 Penhasi et al.
[45] Feb. 20, 1973 [54] PROCESS FOR THE SEQUENTIAL DEGRADATION OF PEPTIDE CHAINS [75] Inventors: Harry A. Penhasi; Henry Meier,
both of Cupertino, Calif.
[52] U.S. Cl. ..23/230 R [51] Int. Cl. ..G0ln 31/00, GOln 31/06 [58] Field of Search ..23/230 R, 230 B, 253 R;
[56] References Cited UNITED STATES PATENTS 3,536,450 10/1970 Dusetal. .......23/253 OTHER PUBLICATIONS Badger and Banchero, Introduction to Chemical Engineering (1955), pp. 469, 497, 499.
Primary Examiner-Morris O. Wolk Assistant Examiner-D. G. Millman Attorney-Stanley M. 'leigland and Robert J. Steinmeyer [57] ABSTRACT An improvement in the process of sequentially degrading peptide chains in a reaction vessel by successive coupling and cleavage reaction steps, wherein the vessel is subjected to a vacuum purging operation between the reaction steps to remove undesired materials. The improvement involves flushing the vessel with a stream of nitrogen gas while it is under vacuum whereby the undesired materials are more rapidly and more completely removed from the vessel and the cumulative yield of the process is increased.
5 Claims, 2 Drawing Figures bal PATENTED FEB 2 01973 Fig SHEET F 2 Len 1h Of Insulin 8 I5 3O 4O 5O 6O 7O Cycle Number INVENTORS HARRY A. PENHASI HENRY MEIER PATENTEUFIEBZOIQH SHEET 2 BF 2 Phe Thr Lys Leu His CM- Ser Leu Phe Asn Ala INVENTORS. HARRY A. PENHASI BY HENRY MEIER Cycles Fig.2
PROCESS FOR THE SEQUENTIAL DEGRADATION OF PEPTIDE CHAINS BACKGROUND OF THE INVENTION This invention relates to an improved method for performing the sequential degradation of peptide chains for purposes of analyzing the constituents of the chain.
It is important to analyze the amino acid sequence of proteins and peptides in order to understand their biological functions. For example, the function of insulin is dependent upon its particular amino acid sequence. A variety of techniques may be used to determine the linear order of amino acids. One of the most important sequential methods now being used is called the Edman process. The Edman process was originally described in Acta Chem. Scand. 4, 283 (1950). Three more recent articles describe the form of its current use, Blomback et al., Human Fibrinopeptides Isolation, Characterization, and Structure, Biochem, Biophys. Acta, 115 (1966) 371-396; Edman and Begg, A Protein Sequenator, European J. Biochem. l (1967) 80-91; Niall et al., The Amino Acid Sequence of Porcine Thyrocalciton, Proc. of the National Academy of Sciences, Vol. 59 No. 4, pp. l,32l-1,328, April 1968; Niall, Sequential Analysis of Proteins and Peptides Fractions No. 2, pp. l-lO (1969). Briefly, as discussed in the last article and as shown inside the cover of the Fractions publication, the Edman sequential degradation processes involve three stages: coupling, cleavage, and conversion. In the coupling stage phenyl isothiocyanate (PITC) reacts with the N-terminal 01 amino group of the peptide to form the phenylthiocarbamyl (PTC) derivative. The
pH is normally maintained at between 9 and 10 and preferably between 9 and 9.5 for the coupling reaction. ln the cleavage step anhydrous acid is used to cleave the PTC derivative, i.e., the anilinothiazolinone (ATZ). After extraction of the thiazolinone the residual peptide is ready for the next cycle of coupling and cleavage reactions. Aqueous acid is used to convert the thiazolinone to the phenylthiohydantoin (PTl-I) which may be analyzed in an appropriate manner such as by chromatography.
At the end of the coupling step the excess PITC and the organic constituents of the coupling buffer, which is used to maintain the desired environment (such as pH) for the coupling reaction, are removed by extraction with benzene. Certain breakdown products of PITC such as aniline and phenylthiourea are also removed. Diphenylthiourea, another side product, is poorly soluble in benzene but may be removed by further extraction with a more polar organic solvent such as ethyl or butyl acetate. The water from the coupling buffer must also be removed, for example by lyophilization. The reaction vessel is then subjected to a vacuum purging operation to remove any remaining undesired materials and the dried protein as its PTC derivative is then ready for cleavage.
During the cleavage operation the protein is dissolved in anhydrous acid, for example trifluoroacetic acid. Thus there is a strong shift in pH from basic for the coupling reaction to acidic for the cleavage reaction. The acid is then evaporated and the cleaved thiazolinone derivative is separated from the residual peptide by extraction with butyl chloride or ethylene dichloride. After evaporation of residual solvent and a further vacuum purging operation, the peptide or protein, now one residue shorter, is ready for the next cycle.
In the typical apparatus used to perform the coupling and cleavage steps, each series constituting a cycle, the steps may be broken down into seven stages. A typical apparatus for accomplishing this is the Beckman Protein Peptide Sequencer. The first of the seven stages is the coupling to the protein. This is followed by two wash stages giving precipitation of the sample along with a primary extraction of excess reagents and a secondary extraction of excess reagents. The cleavage stage, cleaving the amino acid residue on the end of the protein comes next. This is followed by a first extraction which extracts the cleaved amino acid residue. If desired, to be sure that all of the amino acid residue has been cleaved, a second cleavage step may be used followed by a second extraction step.
In the Edman or Beckman type instrument all the functions are performed in a continuously spinning cylindrical cup which comprises the reaction vessel. The protein spreads as a thin film over the surface of the lower half of the cup. Reagents are pumped in so as to flow over the proteins. The movement of one thin film over another is almost ideal for rapid solution, reaction, and extraction maneuvers. Solvent extractions are carried out by allowing the liquid to flow up over the surface of the dried protein to the top of the cup where it reaches a groove in the surface of the cup and can be scooped off. The spinning of the cup also helps to reduce the tendency of material to boil during evaporation of liquids. A nonvolatile base such as that known in the trade as Quadrol, a registered trademark of Wyandot Chemical Corporation, may be used in the coupling buffer and an acid of low volatility is used for cleavage. Thus heptafluoro-butyric acid may be used instead of its more volatile analog trifluoroacetic acid. Quadrol is N,N,N,N tetrakis [2-hydroxypropyl] ethylene diamine. A typical Quadrol buffer is a one molar solution of Quadrol in a 50-50 mixture of propanol and water with the pH adjusted to 9.0 by trifluroacetic acid.
One continuing problem has been loss of sample during the various extraction and reaction steps of each cycle. It is difficult to remove all undesired materials completely from the reaction vessel at the end of each step. Remaining materials can adversely affect the reactions in the next step of the process. For example the carry-over of residual amounts of water and base from the coupling step can interfere with the cleavage reaction which must occur in an anhydrous acid environment. The results of this interference and altered environment show up in overlap and reduced yield of subsequent cycles. At the end of each cycle the amount of sample remaining for the succeeding cycles is lower than desired. This influences the number of cycles that can be carried out. Since proteins or peptides can be many cycles or amino acid residues in length, it is desired to be able to perform as many cycles as possible. A so-called 1 percent confidence level is normally used. That is, the yield of material from any given cycle to produce accurate or usable results must be at least 1 percent of the yield from the initial cycle over and above the background. With respect to the degradation of peptides, this has usually placed a limit of to cycles upon the degradation sequence. The reason for this is that the gradual fall in yield is accompanied by a gradual appearance of a background of other PTI-l amino acids due mainly to incomplete reactions, physical loss in washing, and random cleavages along the peptide chain. This combination of a falling signal with a rising noise level makes identification impossible after a finite number of cycles.
SUMMARY OF THE INVENTION It is an object of the instant invention to provide an improved process for sequentially degrading peptide chains whereby undesired materials are more rapidly and more completely removed from the reaction vessel at the end of each stage of the process so that the yield of the process is increased and the sequence of degradation cycles can be extended. It is an advantage of the instant invention that the number of cycles through which the degradation sequence may be car ried out before the 1 percent yield level, as explained previously, is reached is significantly increased.
The process of the instant invention involves sequentially degrading peptide chains in a reaction vessel by successive coupling and cleavage reaction steps constituting a cycle. The vessel is subjected to a vacuum purging operation between reaction steps to remove undesired materials. The improvement comprises flushing the vessel with a stream of nitrogen gas while it is under vacuum so that the undesired materials are removed more rapidly and more completely from the vessel. As a result the cumulative yield of the process and the yield per cycle are both increased.
Typically the vacuum level is from about 450 to about 550 microns of mercury and the flow rate of nitrogen is about 100 cubic centimeters per minute. The preferred vacuum level is about 500 microns of mercury. When the instant invention is practiced a cycle will take about 70 to 90 minutes. The nitrogen stream desirably passes over substantially the entire surface area of the material in the reaction vessel.
BRIEF DESCRIPTION OF THE DRAWING Other objects and advantages of this invention will be apparent from the description taken in conjunction with the accompanying drawing.
FIG. 1 is a graph comparing the yield per cycle in typical sequential degradations of insulin B with and without the practice of the instant invention.
FIG. 2 is a graph showing the identification and yield of each peptide residue for insulin B as determined utilizing the instant invention.
DETAILED DESCRIPTION A series of comparative sequential degradations were performed using the Beckman Protein Peptide Sequencer as described in the Niall article in Fractions mentioned above. All tests were performed upon insulin chain B. Insulin chain B is known to have residues and is the larger of the two subunits of insulin. It may be described as having a phenylalanine amino terminal and two cystane residues which form bridges with insulin chain A. Heptafluorobutyric acid was used in the cleavage steps in all runs. Phenylisothiocyanate was used in the coupling reactions and different buffers were utilized. Table 1 sets forth the operating conditions and repetitive yields with the weighted average yield and Table 11 sets forth the yields per cycle as a percentage of the first cycle yield in the compared experiments.
TABLE I Insulin B exp. N bufproinitial repetitive yields-% wtd. ave. no. flush fer gram yield 2-12 6-ll ll-lS l2-l8 yield 38 Yes A l2 38.0% 87.1 89.5 95 93.2 90.4 71 Yes C l2 49.0 90 90 89 92.5 v 90 75 No C 13 46.0 84 SI 86 87 34.5 81 No E 14 47.2 89 83 90 87 82 No C l4 43.2 87 84 89 TABLE II Yield Per Cycle-% of First Cycle Yield exp. no. 2 6 ll l2 l5 18 23 28 38 92.2 43.7245 23.5 20.3 15.3 6.9 5.1 71 93 49.7293 32.8 l8.7 19.2 4.7 1.3 75 39.2 43.2l5.4 15.7 3.6 7.l l.5 Bl 90.2 50519.6 30.0 12.9 10.5 2.1 82 89.7 4l.4l6.8 23.4 l3.5 10.4 L9
Buffer A contained three parts of triethylamine to 55 parts of proponol and 42 parts of water and was used to set the pH at 9.7. Buffer C contained 12 parts of dimethylallylamine to 44 parts of proponol and 44 parts of water giving apH during the coupling operation of 10.85. Buffer E contained 12 parts of dimethylallyamine to 44 parts of pyridine and 44 parts of water giving a pH of 9.5.
Benzene was used as the wash liquid in the extraction operation after the coupling step. Butyl chloride was used as a secondary extractant wash liquid. Following the cleavage operation the cleaved thiazolinone derivative was separated from the residual peptide by extraction with butyl chloride.
The conversion operations were performed separately and the various groups identified in accordance with standard chromatographic techniques. The conversion and identification operations form no part of the instant invention and need not be discussed further here.
One hundred cc per minute of nitrogen with 450 to 550 desirably 500 microns of mercury vacuum measured at the pump seem to work quite well and was generally used in the tests for the nitrogen flushing operation.
As shown in the Table, three different programs were used, programs 12, 13, and 14. Program 12 involved a cycle having a single cleavage step with nitrogen flushing, the cycle lasting approximately 76 minutes. Program 13 also involved a single cleavage with no nitrogen flushing, but with the vacuum purging operation between reaction steps, the cycle taking approximately 97 minutes, a difference of 21 minutes. Program 14 involved a single cleavage with an optional double cleavage being permitted. The program could be run both with or without nitrogen flushing between reaction steps. When no nitrogen flushing was used the reaction cycle averaged approximately 96 minutes. When nitrogen flushing was used a reaction cycle normally took about 80 minutes, a difference of 16 minutes. In the experiments shown in the Table, program 14 was run without nitrogen flushing.
FIG. 1 is a graph comparing weighted yields per cycle as a percent of the initial yields. Line 3 represents experiment 75, which was performed without nitrogen flushing, hits the 1 percent yield level at the end of the twenty-ninth cycle. This is barely satisfactory for an analysis of insulin chain B which has thirty residues. Line 2 represents experiments 81 and 82, which were performed without nitrogen flushing but with a double cleavage, showed somewhat better results in that a complete analysis of insulin B was possible. The l percent level is reached by extrapolation at about cycle 35, which indicates a peptide chain having 35 residues could be successfully analyzed. Line 1 represents the results of experiments 38 and 71, both of which used nitrogen flushing in the vacuum purging steps between reaction steps, shows a 4 A percent yield at the end of insulin chain B and extrapolates to the 1 percent level at 45 cycles. This indicates a peptide chain of 45 residues could be successfully analyzed utilizing the instant invention, a significant improvement over the comparable process without nitrogen flushing.
The runs with nitrogen flushing had a weighted average yield of 90 percent or better, significantly superior to the comparable run 75, which had a weighted average yield of 84.5 percent. The experiments utilizing a double cleavage step showed improved yields over the similar program with a single cleavage but did not attain the results of the single cleavage program with nitrogen flushing. It is further significant that the repetitive or cumulative yields for experiments 38 and 71, the experiments which utilized nitrogen flushing in the vacuum purging operation, were significantly higher than those of experiment 75, which used no nitrogen flushing, and were generally higher than those of experiments 81 and 82, which used a double cleavage operation with no nitrogen flushing.
FIG. 2 is a graph showing the identification and yield of each peptide residue for insulin B as determined in experiment 38. The amount of each residue analyzed is plotted as the log mole of the phenylthiohydantoin. The name of each residue and its abbreviation is shown in Table III.
Glutamic Acid Glu Proline Pro The quantity of each residue is plotted with the exception of His and Arg. The presence of these residues could be determined qualitatively but the amount could not be determined quantitatively utilizing the standard chromatographic techniques. The graph illustrates that, utilizing this invention, ade uate quantities of all residues were present for the den ification of the residues. This further illustrates, the utility of the instant invention.
In all steps, the nitrogen stream was passed into the cup-shaped reaction vessel along the axis thereof so that the nitrogen stream passed over substantially the entire surface area of the material in the reaction vessel, which in the description of the sequencer operation was stated to be present as a thin film over the surface of the lower half of the cup or reaction vessel.
It is believed the use of a nitrogen flushing operation in connection with the vacuum purging made possible the more complete removal of the undesired materials at the end of each reaction step. This permitted the succeeding reaction step to proceed under the desired conditions with a minimum deviation therefrom and with a minimum of interference present to cause background reactions. As a result the rise of noise level and fall of signal was less for each cycle and identification of residues was possible for more cycles.
It will be appreciated that the foregoing is a description of exemplary processes according to the instant invention. This is for illustrative purposes only, and the instant invention is not to be limited thereby, but only by the claims wherein:
What is claimed is:
1. In the process of sequentially degrading peptide chains in a reaction vessel by successive coupling and cleavage reaction steps, constituting a cycle, wherein the vessel is subjected to a vacuum purging operation between reaction steps to remove undesired materials, the improvement which comprises flushing the vessel with a stream of nitrogen gas while it is under vacuum whereby the undesired materials are more rapidly and more completely removed from the vessel and the eumulative yield of the process is increased.
2. The process of claim 1 wherein each reaction cycle takes from about to about minutes.
3. The process of claim 1 wherein the nitrogen stream passes over substantially the entire surface area of the material in the reaction vessel.
4. The process of claim 1 wherein the vacuum level is from about 450 to about 550 microns of mercury and the flow rate of nitrogen is about cubic centimeters per minute.
5. The process of claim 4 wherein the vacuum level is about 500 microns of mercury.
Patent Citations
Non-Patent Citations
Referenced by
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text:
yes i would recomend this book ,it is veryuplifting and gives you peace of mind
when she talks about her finding her gift
yes i couldnt stop listening
i am buying this book hardback for christmas gifts for my sisters.
i would recommend this story because it keeps you wanting more.
i have been to sugar creek and just trying to in-vision the places with the places in the story is exciting. i had to cry in some parts i don't want to reveal the story.
if you like reading the amish stories this series is the best i have read yet.
couldn't keep up with all the jumping around . not at all what i expected. i thought this was about wedding planners but couldn't figure this out.
i always look forward to debbie's cedar cove series. would have liked to do them in order but all and all good book
i would like to hear the sequels to this book. kinda left you hanging.want more
very confusing hard to stay with the plot. not what I expected and the nariator was hard to follow with the english accent.
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Kukla's Korner Hockey
Category: NHL-Talk
Just Talk
by Paul on 10/14/07 at 10:11 AM ET
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from Bruce Garrioch of the Ottawa Sun,
One GM on the lack of trades: "Nobody makes them anymore. You've got to give up cash to get rid of cash and it's usually not worth the risk. I talk, and talk, and talk, and it usually leads nowhere."
more... at least you can read about all the trade talk…
Filed in: NHL Talk, | KK Hockey | Permalink
The Portzline Evaluation Formula
by Alanah McGinley on 10/14/07 at 07:26 AM ET
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From Aaron Portzline at the Columbus Dispatch,
Warning: Once you've read this and studied all the numbers, the rest of the NHL's regular season will be a mere formality. The Stanley Cup playoffs, too, for that matter.
The Portzline Evaluation Formula was dreamed up nearly three years ago in the hallucinating cold of Edmonton and fine-tuned this summer in Toronto with the help of a sudsy friend named Alexander Keith.
It is a gloriously subjective and simple way to evaluate each of the 30 NHL teams and, thus, predict how the 2007-08 season will play out.
We're publishing it now (yes, it's copyrighted) so we can say 'We told you so' next June … when the Detroit Red Wings beat the Ottawa Senators in the Stanley Cup Finals.
Filed in: NHL Talk, | KK Hockey | Permalink
Campbell On Jesse Boulerice Suspension
by Paul on 10/13/07 at 11:04 AM ET
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note 1:22pm, I missed my morning coffee earlier today and forgot to note this was from a tele-conference Campbell did yesterday.
Q. So often in these things we hear about repeat offender things, that each suspension thereafter is harsher. Now we've had two against a team in a short period of time. Is there any provision where teams can now be held responsible for their players' actions as well?
COLIN CAMPBELL: There's nothing formal that holds a team responsible. I guess if you really look at the issues they have to deal with, their roster situation, they have to deal with paying the player and with other aspects that come with losing two players that they're paying. But there's nothing formal that punishes the team for the number of players who are suspended.
Filed in: NHL Teams, Philadelphia Flyers, NHL Talk, Cheap Hits, | KK Hockey | Permalink
Tags: colin+campbell, jesse+boulerice, philadelphia+flyers, suspensions
Will NHLPA Distance Itself From The League
by Paul on 10/13/07 at 08:34 AM ET
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from Rick Wethead of the Toronto Star
During Saskin's tenure at the union, emails reveal that NHL chief legal officer and deputy commissioner Bill Daly passed on to Saskin several suggestions about which players and union staff might be conspiring against him. Other emails reveal Saskin had become especially close to Daly and Gary Bettman after his hiring.
In advance of a meeting in Russia in autumn 2006, Saskin emailed the NHL commissioner: "Looks like Bill is getting the Moscow trip in September; I may send Chelios."
In a response the same day Bettman deadpanned: "With a one-way ticket (cc not bd)?"
read on... getting ugly again folks…
Filed in: NHL Talk, NHL Business of Hockey, NHLPA, | KK Hockey | Permalink
Tags: bill+daly, nhlpa, ted+saskin
Vanbiesbrouck Inducted into U.S. Hockey Hall of Fame
by Alanah McGinley on 10/13/07 at 02:26 AM ET
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From the AP via Sports Illustrated,
John Vanbiesbrouck entered the U.S. Hockey Hall of Fame on Friday night as the NHL's winningest American-born goalie.
"One statistic you don't know is that I had almost an equal number of losses,'' the former Rangers star said. "That meant for a lot of cold, dark rides in New York City. It's in those times that you grow, gain courage and ask the deepest questions.''
Vanbiesbrouck retired in 2002 with a 374-346-119 record. He won the Vezina Trophy as the NHL's best goalie in 1986.
Filed in: NHL Talk, | KK Hockey | Permalink
Tags: goalies, john+vanbiesbrouck
7 Rookies Off to Hot Starts
by Alanah McGinley on 10/13/07 at 02:05 AM ET
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From Craig Custance at the AJC,
This season's crop of rookies may the deepest since the lockout. There's not another Crosby, but then again, when will there ever be? But there's a young goalie in Montreal who people are already comparing to Patrick Roy and the Blackhawks have a player providing hope for a bright future. In honor of Thrashers rookie defenseman Mark Popovic, who wears No. 7, here are the top seven rookies already making an impact:
1. NICKLAS BACKSTROM, Forward, Capitals
Filed in: NHL Teams, NHL Talk, | KK Hockey | Permalink
Tags: prospects
Bettman Covers All The Bases
by Paul on 10/12/07 at 06:32 PM ET
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from Evan Weiner at MCN Sports,
The National Hockey League's regular season is just two weeks old and it is far too early to analyze any trends such as attendance and TV ratings, there have been a few interesting developments so far. The National Hockey League will have a hockey channel available to a good number of cable TV subscribers in the United States and it appears that the league's venture into London, staging two regular season games between the defending Stanley Cup champion Anaheim Ducks and the Los Angeles Kings, was a success.
There is also some talk that the league could open the 2008-09 season in Prague, The Czech Republic.
continued and the topics are many. Wings fans, Bettman even questions Jimmy D. and his statement about Las Vegas and the Wings moving to the East…
Filed in: NHL Talk, | KK Hockey | Permalink
Tags: gary+bettman
Grand Opening in Manhattan
by Alanah McGinley on 10/12/07 at 06:19 PM ET
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The new NHL—Powered by Reebok store had its official grand opening today.
The grand opening of the first-ever NHL-themed store in New York brought thousands of fans to the new facility, as well as some hockey legends like Glenn Anderson, Bruce Driver, Ken Daneyko, Rick Middleton, Eric Cairns, Butch Goring, former referee Paul Stewart, and of course, the biggest star of them all, the Stanley Cup.
Photo gallery has some great shots here.
Filed in: NHL Talk, NHL Business of Hockey, | KK Hockey | Permalink
Tags: merchandising
Take The Suspensions A Step Further
by Paul on 10/12/07 at 06:09 PM ET
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from Eric Duhatschek of the Globe and Mail,
...There are, however, signs, that the NHL is trying to change that mindset, one suspension at a time — the Boulerice suspension ties with Chris Simon for the longest in history, relating to an on-ice incident.
But the league could fast-track the process by extending the discipline to the respective teams as well. Fines to the club, suspensions for the coach - those sorts of penalties might encourage teams to think twice about employing players who haven't yet figured out that the culture of head-hunting is going the way of the dodo.
The simplest solution might even be the least complicated — simply prevent a team from replacing a suspended player in the line-up.
more and other NHL bits too in Eric's weekly NHL roundup…
Filed in: NHL Talk, Cheap Hits, | KK Hockey | Permalink
Tags: suspensions
Tootoo Went For The Head
by Paul on 10/12/07 at 02:07 PM ET
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from Bob McKenzie at TSN,
Jordin Tootoo of the Nasvhille Predators takes a run at Phoenix rookie Daniel Winnik – and it says here Tootoo was targeting Winnik's head – and it's Coyote rookie Craig Weller who gets the match penalty for intent to injure on the play, because Weller clotheslined Tootoo after the hit….
But it's the Tootoo hit that should be the real concern.
If ever there were a hit where it looked like a player was intentionally trying to hit someone in the head, this was it.
more... I haven't seen the video of the play from last night, if anyone has a link, feel free to post it.
update 12:14pm, Thanks to KK member bcrt2000, we have a link…
Filed in: NHL Teams, d, Nashville Predators, NHL Talk, Cheap Hits, | KK Hockey | Permalink
Tags: jordin+tootoo, nashville+predators
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About Kukla's Korner Hockey
Paul Kukla founded Kukla's Korner in 2005 and the site has since become the must-read site on the 'net for all the latest happenings around the NHL.
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Catch up on all of Yuma's epic duels with full episodes and clips here


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Vacation Preparation: Is Your House Ready for your Summer Trip?
You're all set to leave on your summer trip – but is your house as vacation-ready? Here are some helpful tips to prepare your home before your departure so you can travel worry-free!
You're all set to leave on your summer trip – but is your house as vacation-ready? Here are some helpful tips to prepare your home before your departure so you can travel worry-free!
One of the most important insurance policies you can buy is the personal umbrella policy, but many people are unaware they need one.
On Saturday, June1 we held a rummage sale at Insure Forward in Bemidji with 100% of the proceeds benefiting Forward Foundation. The rummage sale went very well and was a great community event to help spread the word about our cause!
Adapted from the article originally published by the FDIC, available at http://1.usa.gov/12Nw2Ah
If you're interested in trying out a new banking technology but you're not sure what's available or how it would be helpful for you, read on. This article offers an overview today's mobile services - and many of them and free!
Bank and Insure Forward is pleased to announce the winners of their 2013 Education Scholarships. Bank/Insure Forward's scholarship program is based upon individual student grades, participation in extracurricular activates and community involvement.
ACUITY recently recognized Insure Forward for their 25th anniversary of their business relationship.
You have a great idea for a business. You've also heard common statistics such as those from the Small Business Administration that say only 56% of all small businesses are still around at their five-year mark.
"Everyone lives in a flood zone" and 9 other helpful tips, courtesy FEMA, on flood insurance. Still have questions after reading? Contact an agent today!
The University of Minnesota Crookston will auction off a framed autographed Zach Parise Minnesota Wild jersey in a timed raffle during their annual UMC Teambacker "Fun Nite" held Friday, April 26 at the Crookston Eagles.
Adapted from the article originally published by the FDIC, available at http://1.usa.gov/Zno8L3
Tax season is wrapping up. Now, you're left with a refund (if you're lucky) and piles of documents you're unsure what to do with.
Technological devices such as tablets and cell phones have made it easy to stay connected every minute of every day. Bank customers benefit because they can use mobile devices to view accounts whenever they want by mobile banking.
Bank Forward believes in their community's future leaders and wants to help them attain their goals by rewarding them for their accomplishments in academics, activities, as well as their community involvement.
Are you getting a big fat refund this year, or writing a check to the IRS for taxes owed? We asked Bank and Insure Forward employees in Fargo, Grand Forks and New Rockford what they are doing with their tax refund.
The school year is wrapping up but learning is something that never ends. We asked Cooperstown and Wimbledon employees, "What financial life lessons did you (will you) give your own kids?" Here's food for thought from the Bank Forward pros.
What you are required to do when you hire a subcontractor
Reprinted by permission of SFM
Hiring an uninsured subcontractor poses a significant liability to your organization. This liability can affect your workers compensation premium substantially. This notice explains what you must do to avoid unexpected additions to your premium.
If you're considering a national tax preparer that promises faster refunds by offering Refund Anticipation checks (RACs), get the full story.
You have likely seen or heard advertisements on billboard, TV or radio for "free tax filing" of 1040EZ returns this season from certain tax preparation companies.
Beware! We think it's important to know exactly what this means to avoid unanticipated tax preparation costs and fees associated with financial products that promise fast refunds, such as Refund Anticipation Checks.
Insure Forward, Grand Forks, announces that Alicia Driscoll has been promoted to Personal Lines Sales Agent.
If your employee uses his or her own personal vehicle for work-related duties, what happens if he or she has an accident? Generally, your employee's auto insurance company would pay for any damage, but if their coverage is insufficient, who pays?
Bank Forward, Grand Forks, announces that Brady Trenbeath has been promoted to Market President for the Grand Forks Bank Forward locations.


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Review: Rowland mixes R&B, Euro sound on new CD
Kelly Rowland, "Here I Am" (Universal Motown)
Ever since her heyday with Destiny's Child, Kelly Rowland has struggled to define her solo singing career, unlike former bandmate Beyonce, arguably pop's ultimate superstar.
Rowland's first two albums showed promise, but were subpar on the whole. Now with her latest release, "Here I Am," she makes another attempt to show she has finally found her musical niche.
Rowland's latest album is filled mostly with R&B melodies and some European uptempo dance tracks. She displays confidence in her self-worth with a few songs that should compel you to press replay. One in particular is her chart-topping single "Motivation," featuring Lil Wayne, which proves that she has the potential to fully carry herself as a solo artist.
The sexy vocalist also has a host of producers, including Rodney Jerkins, Jim Jonsin, RedOne and David Guetta. But the top-notch producers are not enough to elevate Rowland's star status on this album. Most of the 10 tracks struggle to meet the mediocre mark and simply do not measure up to the expectations brought by her infectious "Motivation."
The closest Rowland gets to good is on "Feeling Me Right Now." She's bold in her infatuation with herself on the mid-tempo track, claiming she can escort herself home without the presence of a man after a night out at the club. She sings on the piano-driven track, "I fell in love in the club tonight/I'm so feelin' me right now/I wanna take her home, take her home/'cause I know I'll love her right."
The "Feeling Me" track, along with "Keep It Between Us," "All of the Night" featuring Rico Love, and "Turn It Up," fit Rowland's strong and likable voice. However, she falters on the Guetta-produced "Commander," which sounds more like a track that should have been made for Rihanna rather than Rowland.
Unfortunately, seven years after Destiny's Child's last album, Rowland is still trying to find the route to her own musical destination.
CHECK OUT THIS TRACK: On "Keep It Between Us," Rowland croons impressively on the buoyant ballad for a satisfying listen.
Follow Jonathan Landrum Jr. on Twitter at http://www.twitter.com/mrlandrum31
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Cupcakes I made for my dad for fathers day some of his fave things.
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Oregon carbon tax measure set for next step — if there is one
- Andy Giegerich
- Digital Managing Editor- Portland Business Journal
- Email | Twitter | Google+
A "gut-and-stuff" strategy designed to keep a key sustainability measure alive in Oregon's legislative chambers has apparently worked.
However, with time running out on the state's 2013 legislative session, it's not clear whether the carbon tax-related initiative Senate Bill 306 will get full Oregon House consideration.
The Oregon Senate quietly passed the bill this week in between debate over the state's budget. SB 306 directs Oregon's revenue officer to prepare reports on the feasibility of a statewide "clean air fee or tax."
The measure passed the Senate by an 18-12 vote on Wednesday. The measure immediately went to House Speaker Tina Kotek who quickly turned it back over to the joint Ways and Means Committee.
The "gut-and-stuff" move put two House measures into one bill, the dead-on-arrival corporate kicker measure, in an effort to keep both issues alive.
A Portland State University study released earlier this year found that a carbon tax could deliver billions to the state's budget.
Andy is the Portland Business Journal's digital managing editor, overseeing the daily digital news operation.
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Orlando – James Rode is the new artistic director and conductor of Orlando Gay Chorus.
OGC celebrates 25 years this year and Rode is ready to take on this new challenge.
"It's exciting and a new adventure to kind of jump on," Rode said.
OGC's President of the Board of Directors Karl Haglund made the announcement Feb. 16.


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Forbidden love and secret hate
Tiffany, purpleinopp GardenWeb, Z8b Opp, AL(8B AL)October 13, 2011
What does everyone else seem to love that you hate?
What does everyone else hate that you secretly love?
I can't stand stella d'oro day lilies or moonbeam coreopsis. Nandina and oleander are totally boring to me.
Violets and weedy grew-by-themselves morning glories make me happy (except during the few hours it takes to remove all of the dead vines from the fence.)


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Swimming: Willmar dips Montevideo
WILLMAR -- The Willmar Cardinals outswam visiting Montevideo 102-79 in a non-conference swimming and diving dual on Tuesday.
A.J. Derouin and Michael McLain, both Willmar freshmen, each won two individual events. Ross Swartz also was a double winner.
Willmar is 2-0 after opening the season with a win over CLC rival Sauk Rapids.
Willmar 102, Montevideo 79
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A winter storm has many upsides: snow days, skiing, and the chance to follow deer back to the tree where they roost. Experience some winter magic with this GrouponLive deal.
The Deal
- One-day tickets to see Essent ISU World Cup Speed Skating
- Where: Utah Olympic Oval
- Seating: General admission
- Door time: One hour before that day's event start time
- Ticket values include all fees.

Tickets and Dates
- $10 for one-day admission for four people (a $20 value)
- $20 for one-day admission for eight people (a $40 value)

For either option, choose one of the following dates:
- Friday, November 15, at 2 p.m.
- Saturday, November 16, at 1:30 p.m.
- Sunday, November 17, at 1:30 p.m.

Essent ISU World Cup Speed Skating
More than 200 top skaters from 25 countries gather at the Utah Olympic Oval on November 15–17 for a World Cup event with serious ramifications for the upcoming Olympic Games. Men and women from around the world square off in long-track distance races, vying for the fastest time while avoiding holes in the ice carved by local fishermen. On Saturday, fans congregate before the races for a Wild West-themed gathering with skating activities, bounce houses for children, and live music and entertainment.
Utah Olympic Oval
It's hard not to notice that the ice of the 400-meter skating oval is steeped in history. Eight world speed-skating records and ten Olympic records were set on the Utah Olympic Oval's ice during the 2002 Winter Games. Now, visitors can glide over the same surface during public skating sessions while gazing at the record times immortalized on the walls. Inside the comfortable, 65-degree facility, guests can also race around the perimeter of the ice on the running track, cheer on hockey players at two rinks, learn to skate during lessons, or send stones hurtling toward their friends during cosmic curling games.
Utah Olympic Oval
3419 Olympic Parkway
Park City, UT 84098
+14356584200Get Directions